Zinc finger nuclease for the cftr gene and methods of use thereof

ABSTRACT

The present invention provides new zinc finger proteins and zinc finger nuclease (ZFNs) that find particular using in repairing the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

The present application is a continuation-in-part of PCT ApplicationPCT/US2009/040617, which claims benefit of priority to U.S. ProvisionalApplication Ser. No. 61/124,297, filed Apr. 16, 2008, the entirecontents of both applications being hereby incorporated by reference.

This invention was made with government support under 1R21HL91808-01awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates to the fields of molecular biology andmedicine. More particularly, it relates to zinc finger nucleases andtheir use in treating cystic fibrosis.

II. Related Art

A. Cystic Fibrosis

Cystic fibrosis, or CF, is an inherited disease of epithelia-linedorgans, including the glands that make mucus and sweat. “Inherited”means that the disease is passed through the genes from parents tochildren. People who have CF inherit two faulty cystic fibrosistransmembrane conductance regulator (CFTR) genes, one from each parent.CF mostly affects the lungs, pancreas, liver, intestines, sinuses, andsex organs. The symptoms and severity of CF vary from person toperson—some people who have CF have serious lung and digestive problems,while others have more mild disease that does not show up until they areadolescents or adults. The symptoms and severity of CF also vary overtime.

The disease manifests because if one has CF, mucus becomes thick andsticky. In the lungs, airway innate immunity is impaired, mucus buildsup and blocks airways. The buildup of mucus makes it easy for bacteriato grow, which leads to repeated, serious lung infections. Over time,these infections can severely damage lungs. The abnormal secretions alsocan block ducts in the pancreas. As a result, the digestive enzymes thatyour pancreas makes cannot reach your small intestine, causing vitamindeficiency and malnutrition because nutrients leave the body unused. Italso can cause bulky stools, intestinal gas, a swollen belly frompartial or complete intestinal obstruction, and pain or discomfort. CFalso causes your sweat to become very salty and, as a result, your bodyloses large amounts of salt when you sweat. This can upset the balanceof minerals in your blood and cause a number of health problem,including dehydration, increased heart rate, tiredness, weakness,decreased blood pressure, heat stroke, and, rarely, death.

As treatments for CF continue to improve, so does life expectancy forthose who have the disease. Today, some people who have CF are livinginto their thirties, forties, fifties, or older. However, CF remains theleading genetic cause of premature death in the United States. As such,improved methods of treating CF are in great need.

B. Zinc Finger Nucleases

Zinc fingers are among the most common DNA binding motifs found ineukaryotes. It is estimated that there are 500 zinc finger proteinsencoded by the yeast genome and that perhaps 1% of all mammalian genesencode zinc finger containing proteins. These proteins are classifiedaccording to the number and position of the cysteine and histidineresidues available for zinc coordination.

The CCHH class, typified by the Xenopus transcription factor IIIA, isthe largest. These proteins contain two or more fingers in tandemrepeats. In contrast, the steroid receptors contain only cysteineresidues that form two types of zinc-coordinated structures with four(C₄) and five (C₅) cysteines. Another class of zinc fingers contains theCCHC fingers. The CCHC fingers, which are found in Drosophila, and inmammalian and retroviral proteins, display the consensus sequenceC—N₂—C—N₄—H—N₄—C(SEQ ID NO:111). Recently, a novel configuration of CCHCfinger, of the C—N₅—C—N₁₂—H—N₄—C(SEQ ID NO:112) type, was found in theneural zinc finger factor/myelin transcription factor family. Finally,several yeast transcription factors such as GAL4 and CHA4 contain anatypical C₆ zinc finger structure that coordinates two zinc ions. Zincfingers are usually found in multiple copies (up to 37) per protein.These copies can be organized in a tandem array, forming a singlecluster or multiple clusters, or they can be dispersed throughout theprotein.

Zinc finger nucleases (ZFNs) can be used to “rewrite” the sequence of anallele by invoking the homologous recombination machinery to repair thedouble-strand breaks using a supplied DNA fragment as a template. Thehomologous recombination machinery searches for homology between thedamaged chromosome and the extra-chromosomal fragment and copies thesequence of the fragment between the two broken ends of the chromsome,regardless of whether the fragment contains the original sequence. Ifthe subject is homozygous for the target allele, the efficiency of thetechnique is reduced since the undamaged copy of the allele may be usedas a template for repair instead of the supplied fragment.

Custom-designed ZFNs that combine the non-specific cleavage domain (N)of FokI endonuclease with zinc finger proteins (ZFPs) offer a generalway to deliver a site-specific double-strand breaks to the genome, andstimulate local homologous recombination by several orders of magnitude.This makes targeted gene correction or genome editing a viable option inhuman cells. Since ZFN-encoded plasmids can be used to transientlyexpress ZFNs to target a double-strand break to a specific gene locus inhuman cells, they offer an excellent way for targeted delivery of thetherapeutic genes to a pre-selected chromosomal site. The ZFN-encodedplasmid-based approach has the potential to circumvent problemsassociated with viral delivery of therapeutic genes. Alternatively, ZFNpairs can be packaged in a variety of viral vectors to improve deliveryto specific cell types.

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided a zincthree-finger binding domain that targets a nucleotide sequence selectedfrom the group consisting of GTGGAATTA (SEQ ID NO:1) and GAGTGGTTA (SEQID NO:2). The zinc three-finger binding domain may comprise a sequenceselected from wherein one monomer of said dimer comprises a sequenceselected from SEQ ID NOS: 3-5, 6-8, 9-11, 12-14, 15-17, 18-20, 21-23,24-26, 27-29, 30-32, 33-35 and 36-38 for GTGGAATTA (SEQ ID NO:1) andselected from the group consisting of SEQ ID NOS: SEQ ID NOS: 39-41,42-44, 45-47, 48-50, 51-53, 54-56, 57-59, 60-62, 63-65, 66-68, 69-71,72-74, 75-77, 78-80, 81-83, 84-86, 87-89, 90-92, 91-93, 94-96, 97-99,100-102, 103-105, 106-108, and 109-111 for GAGTGGTTA (SEQ ID NO:2). Thezinc three-finger binding domain may be linked to a non-specificnuclease, such as FokI.

In another embodiment, there is provided a zinc three-finger bindingdomain dimer that targets a double-stranded nucleic acid comprising afirst monomer that targets nucleotide sequence GTGGAATTA (SEQ ID NO:1)in one strand and a second monomer that targets nucleotide sequenceGAGTGGTTA (SEQ ID NO:2) in the other strand. A particular monomercombination is SEQ ID NO:116 and SEQ ID NO:117. In a particular form,each monomer of said zinc three-finger binding domain dimer is linked toa non-specific nuclease monomer, such as FokI. The resulting zinc fingernuclease may be homo- or heterodimeric.

In yet another embodiment, there is provided a vector comprising anucleic acid segment encoding a zinc three-finger binding domain thattargets a nucleotide sequence selected from the group consisting ofGTGGAATTA (SEQ ID NO:1) and GAGTGGTTA (SEQ ID NO:2), said nucleic acidunder the control of a promoter operable in a eukaryotic cell. Thevector may further comprise a selectable or screenable marker and or anorigin of replication. The vector may be a viral vector, such as anadenoviral vector, an adeno-associated viral vector, a pox viral vector,a herpes viral vector, a retroviral vector, a lentiviral vector,including an integrase defective lentivirus vector. The vector maycomprises two nucleic acid segments, each encoding a zinc three-fingerbinding domain, one that targets GTGGAATTA (SEQ ID NO:1) and one thattargets GAGTGGTTA (SEQ ID NO:2). In such situations, each of saidnucleic acid segments may be under the control of a separate promoteractive in said eukaryotic cell, or both of said nucleic acid segmentsmay be under the control of a the same promoter. The nucleic acidsegments may be separated by a transcription termination signal,separated by an internal ribosome entry site, or by a picornavirus T2Asequence.

In still yet another embodiment, there is provided a method of promotingrecombination within a CTFR gene in a human cell comprising contactingsaid cell with a first zinc three-finger binding domain that targets anucleotide sequence GTGGAATTA (SEQ ID NO:1) and a second zincthree-finger binding domain that targets a nucleotide sequence GAGTGGTTA(SEQ ID NO:2), wherein each of said first and second zinc three-fingerbinding domains are linked to a non-specific nuclease. The zincthree-finger binding domain may comprise a sequence selected from SEQ IDNOS: 3-5, 6-8, 9-11, 12-14, 15-17, 18-20, 21-23, 24-26, 27-29, 30-32,33-35 and 36-38 for GTGGAATTA (SEQ ID NO:1) and selected from the groupconsisting of SEQ ID NOS: 39-41, 42-44, 45-47, 48-50, 51-53, 54-56,57-59, 60-62, 63-65, 66-68, 69-71, 72-74, 75-77, 78-80, 81-83, 84-86,87-89, 90-92, 91-93, 94-96, 97-99, 100-102, 103-105, 106-108, and109-111 for GAGTGGTTA (SEQ ID NO:2). The human cell may be a lungepithelial cell, and intestinal epithelial cell, a biliary ductepithelial cell, a gall bladder epithelial cell or pancreatic epithelialcell. The lung cell or pancreatic cell may comprise a CFTR gene with aΔF508 mutation.

The lung epithelial cell or pancreatic cell may be located in a livinghuman subject, and contacting may comprise administering said first andsecond zinc three-finger binding domains to lung or pancreatic tissue ofsaid subject. The administration to lung tissue may comprise inhalationor topical instillation. The administration to pancreatic tissue maycomprise injection. Contacting may comprise administering to saidsubject an expression vector comprising a first nucleic acid segmentencoding a first zinc three-finger binding domain that targets GTGGAATTA(SEQ ID NO:1) and a second nucleic acid segment encoding a second zincthree-finger binding domain that targets GAGTGGTTA (SEQ ID NO:2), saidnucleic acids under the control of one or more promoters operable in aeukaryotic cell. The vector may be a viral vector, such as an adenoviralvector, an adeno-associated viral vector, a pox viral vector, a herpesviral vector, a retroviral vector, a lentiviral vector, including anintegrase-defective lentiviral vector. The nucleic acid segments may beunder the control of a separate promoter active in said eukaryotic cellor under the control of a the same promoter. The nucleic acid segmentsmay be separated by a transcription termination signal and/or aninternal ribosome entry site and/or a picornavirus T2A sequence.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions and kits of theinvention can be used to achieve methods of the invention.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-C—OPEN Method for Constructing Zinc-finger Arrays. (FIG. 1A)Schematic overview of OPEN zinc-finger pool construction. Zinc-fingerdomains are represented as spheres and their associated 3 bp subsites asrectangles. The randomized finger in the library is multi-colored. Thestrategy for making finger pools for the middle finger in a three-fingerdomain is illustrated. Note that finger pools for amino- orcarboxy-terminal fingers were obtained by building additional librariesin which finger 1 or finger 3 were randomized, respectively. Details inthe text and Experimental Procedures. (FIG. 1B) Schematic overview ofOPEN selections. Zinc-fingers and associated subsites represented as inFIG. 1A. Details in Experimental Procedures. (FIG. 1C) Schematic of thebacterial two-hybrid (B2H) system. ZFA=zinc-finger array.

FIGS. 2A-D—OPEN ZFNs Engineered to Cleave EGFP Reporter Gene Sequences.(FIG. 2A) Map of sites in EGFP targeted by OPEN selections. (FIG. 2B)DNA-binding activities of zinc-finger arrays made by modular assembly(MA) and OPEN assessed by quantitative B2H assays. The source of modulesused to construct modularly assembled arrays is indicated as Barbas,Sangamo, or Toolgen. Mean fold-activation values (colored bars) andstandard deviations (error bars) obtained from three independent assaysare shown. (FIG. 2C) EGFP-disruption assay for testing ZFN activities inhuman cells. (FIG. 2D) Modularly assembled and OPEN ZFN activitiesassessed using the EGFP-disruption assay. Error bars represent standarddeviations. Single and double asterisks indicate p values <0.05 and<0.01, respectively.

FIGS. 3A-G—Highly efficient mutagenesis of endogenous human and plantgenes by OPEN ZFNs. (FIG. 3A) Map of sites in human VEGF-A, HoxB13, andCFTR targeted by OPEN selections. (FIG. 3B) Schematic of CEL I assay forassaying ZFN-induced mutations. (FIGS. 3C-D) Mutation of the endogenoushuman VEGF-A promoter (FIG. 3C) and HoxB13 gene (FIG. 3D) by OPEN ZFNs.Colored arrows indicate expected CEL I digestion products. Images shownare from representative experiments. (FIGS. 3E-G) Sequences of (FIG. 3E)HoxB13 alleles from human 293 cells transfected with HX587 ZFN pair B,(FIG. 3F) CFTR alleles from human K562 cells transfected with CF877ZFNs, and (FIG. 3G) SuRA alleles from tobacco plants transfected withSR2163 ZFNs. Numbers of each allele identified are shown in parentheses.ZFN recognition sites are in bold orange print.

FIGS. 4A-K—Highly efficient gene targeting of endogenous human loci byOPEN ZFNs. (FIG. 4A) Gene targeting of human VEGF-A and IL2Rγ genes byhomologous recombination with an exogenous “donor construct.” Arrowsindicate primers used for limited-cycle PCR/restriction digest assaydescribed in the text. (FIG. 4B) OPEN ZFNs induce efficient genetargeting at the VEGF-A promoter in human 293 cells. Top part showsrepresentative gel images from limited-cycle PCR/restriction digestassays and bottom part shows gene targeting frequency means (coloredbars) and standard errors (error bars) from multiple experiments. (FIG.4C) OPEN VEGF-A and Sangamo IL2Rγ ZFNs induce efficient gene targetingat endogenous genes in human K562 cells. Data presented as in FIG. 4B.(FIG. 4D) Gene targeting efficiencies of OPEN VEGF-A ZFNs assessed ninedays post-transfection by limited-cycle PCR/restriction digest andSouthern blot assays. (FIG. 4E) Comparison of PCR-based and Southernblot methods for assaying gene targeting efficiencies. Dotted red linerepresents where data points would fall if the two methods wereperfectly concordant. (FIG. 4F) G2 arrest by vinblastine enhances genetargeting by OPEN VEGF-A and Sangamo IL2Rγ ZFNs. Assays performed fourdays post-transfection. Data presented as in FIG. 4B. (FIGS. 4G-I)Sequences of alleles sequenced from human K562 cells transfected with(FIG. 4G) VF2468 ZFNs and donor, (FIG. 4H) VF2471 ZFNs and donor, and(FIG. 4I) IL2Rγ ZFNs and donor. Data are presented as in FIG. 3E. (FIG.4J) Toxicities of OPEN VEGF-A and Sangamo IL2Rγ ZFNs in human K562cells. Means and standard deviations of GFP (green bars) and genetargeting ratios (purple bars) are shown. Single and double asterisksindicate p values <0.05 and <0.01, respectively. (FIG. 4K) Genetargeting efficiencies of OPEN VEGF-A and Sangamo IL2Rγ ZFNs in toxicityexperiments of FIG. 4J. Means and standard deviations are shown ofPCR-based assays performed four days post-transfection.

FIGS. 5A-B—OPEN ZFNs induce permanent alterations of multiple genecopies in human cells. (FIG. 5A) OPEN ZFNs can induce stable alterationof as many as four copies of the VEGF-A gene in a single human cell.Altered clonal cells were genotyped using the limited-cyclePCR/restriction digest assay. Representative results from each genotypeobserved are shown in the bottom panels. Additional details in the text.(FIG. 5B) FISH analysis of wild-type K562 cells and three K562 celllines (modified by VF2468 ZFNs) in which all copies of the VEGF-A genehave undergone a gene targeting event. FISH was performed with a probefor VEGF-A (red) and a control probe for 14q which is present in twocopies per cell (green).

FIG. 6—PCR Strategy for Creating Recombined OPEN Libraries.

FIG. 7—Surveyor nuclease (Cel I) assay autoradiograph. A CF airwayepithelial cell line homozygous for the ΔF508 CFTR mutation wastransduced with a serotype 5 adenovirus vector expressing the indicatedZFN pairs (L1R1, L1R2, etc.). Assay performed 72 hr post transductionwith adenovirus vector. Expected products following ZFN cleavage are 263and 170 nt. Experimental conditions indicated for lanes A-E. Signaldetected by phosphorimager.

FIG. 8—Diagram of a ZFN pair package in an adenovirus vector. ZFN pairsare packaged in a single adenoviral vector. The construct is driven bythe CMV promoter. Heterodimeric FOK1 nucleases are employed. The two ZFNexpression cassettes are joined by an intervening picornavirus T2Asequence. An mCherry reporter cassette is packaged in the E3 region ofthe vector.

FIG. 9—Repair DNA donor template for exon 10 of CFTR. Donor template is1320 nucleotides long. 350 nts of left arm and 970 nts of right arm. Itis packaged in an adenoviral vector but could be delivered by othervectors (viral or non-viral). The 9 nucleotides highlighted in green onthe sequence indicate the regions where the ZFNs bind. In between thetwo ZFN is a 6-nucleotide spacer. The TGTCA fragment contained in thedonor repair template is introduced between the spacer region TGATGAwhich will introduce the unique BspH1-TCATGA only in the donor.

FIG. 10—Sequence of the 2 kb region around the ΔF508 mutation in theCFTR gene. Sequence in exon 10 region is in bold. CTT (outlined font) isabsent in ΔF508 individuals. Lower case is the 790 ZFN binding sites.Double underline is the 877 ZFN binding sites. TGTCA 5 bp insertionbetween TGA/TGA of 877 spacer TGATGA to introduce unique restrictionsite BspH1. Repeat regions are in italics.

FIGS. 11A-B—(FIG. 11A) Depiction of an adenoviral vector expressing theZFN pair targeting the CF877 site as a single expression cassette withan intervening picornavirus T2A sequence. (FIG. 11B) The frequency ofNHEJ increased in a dose-dependent manner from ˜10% at MOI of 50 to ˜29%at MOI of 250 in CFBE4lo-cells.

FIG. 12—Radioactive assay for homologous recombination (HR). K-562 cellswere nucleofected (Amaxa T-16, soln V) with ZFNs and donor DNA or ZFNsonly. The donor template is ˜1.5 kb and has a 5 bp insertion in spacerregion between the ZFN binding site where the ZFNs cleave the genomicDNA. This insertion (TGTCA) creates a unique BspH1 site in the donor.The genomic DNA is harvested 4 days post nucleofection. PCR usingprimers binding outside of the donor template and radiolabeled dNTPs isfollowed by BspH I digestion. In the experiments with ‘+V’, 24 hrs postnucleofection, the cells were treated with vinblastine (0.2 μM) for 16hrs. The presence of fragments at 1136 and 504 bp indicates HR. HR wasconfirmed by DNA sequencing.

FIG. 13—Radioactive assay for homologous recombination (HR) inepithelia. CFBE (CF ΔF508/ΔF508 bronchial epithelial cells) wereco-transduced with adenoviral vectors expressing the ZFN pair (AdZ) andthe homologous recombination donor (AdD) or with AdZ only. The donortemplate is ˜1.5 kb and has a 5 bp insertion in spacer region betweenthe ZFN binding sites. This insertion (TGTCA) creates a unique BspH1site in the donor. The genomic DNA is harvested 4 days posttransduction. PCR using primers binding outside of the donor templateand radiolabeled dNTPs is followed by BspH I digestion. The presence offragments at 1136 and 504 bp indicates HR. Direct DNA sequencing alsodocumented HR in cells receiving AdZ and AdD, but not AdZ alone.

DETAILED DESCRIPTION OF THE INVENTION

Engineered zinc-finger nucleases (ZFNs) are broadly applicable toolsthat have been shown to mediate highly efficient genome modification inDrosophila, C. elegans, plant, and human cells (Alwin et al., 2005;Beumer et al., 2006; Bibikova et al., 2003; Bibikova et al., 2002; Cornuet al., 2008; Lloyd et al., 2005; Lombardo et al., 2007; Miller et al.,2007; Moehle et al., 2007; Morton et al., 2006; Porteus and Baltimore,2003; Szczepek et al., 2007; Urnov et al., 2005; Wright et al., 2005).ZFNs function as dimers with each monomer consisting of a non-specificcleavage domain from the FokI endonuclease fused to an array ofartificial zinc-fingers engineered to bind a target DNA sequence ofinterest (Durai et al., 2005; Porteus and Carroll, 2005). Individualzinc-finger domains bind to 3 bp subsites, and arrays of fingers canrecognize extended 9 or 12 bp sequence targets. ZFNs introducesite-specific, double-stranded DNA breaks (DSBs) that stimulate eitherhighly efficient gene targeting by homologous recombination with anexogenously introduced template (Jasin, 1996) or gene mutation byerror-prone non-homologous endjoining (NHEJ). Absolute rates ofZFN-enhanced gene modification can be as high as 50% (Lombardo et al.,2007).

Lack of access to a rapid and effective zinc-finger engineering platformhas significantly limited the efforts of academic scientists to use andfurther develop ZFN technology. It is striking that in the five yearssince ZFNs were first shown to work in human cells, not one academicgroup has successfully made ZFNs for even a single endogenous mammalianor plant gene (although endogenous Drosophila genes have been modified(Beumer et al., 2006; Bibikova et al., 2003)). The proprietaryzinc-finger engineering platform of the company Sangamo Biosciences isnot freely available to academic scientists and thus remains a “blackbox” with respect to both its implementation and efficacy (Scott, 2005).The published literature describes many different publicly availablezinc-finger engineering methods which can be broadly grouped into twogeneral categories: (1) “modular assembly” methods in which individualfingers with pre-characterized specificities are joined together (Bae etal., 2003; Beerli and Barbas, 2002; Liu et al., 2002; Mandell andBarbas, 2006; Segal, 2002; Segal et al., 2003) or (2) labor-intensiveselection-based methods which require multiple large randomizedlibraries (Greisman and Pabo, 1997; Hurt et al., 2003; Isalan and Choo,2001; Isalan et al., 2001).

Although modular assembly is easy to perform, the inventors haverecently shown that its overall efficacy for making functional ZFN pairsis predicted to be less than 6% (Ramirez et al., 2008) and that evenwhen “successful” it yields ZFNs with low activities and/or hightoxicities (Cornu et al., 2008; Pruett-Miller et al., 2008).Selection-based methods require construction and interrogation ofmultiple, large randomized libraries (typically >10⁸ in size) andtherefore remain intractable for all but a small number of academiclabs. Thus, unlike other broadly accessible technologies like RNAinterference, ZFN-induced genome modification has been utilized by veryfew academic scientists.

The inventors describe here the development and validation of OPEN(Oligomerized Pool ENgineering), a facile and robust platform forengineering customized zinc-finger arrays. OPEN is enabled by theconstruction of a large archive of zinc-finger pools designed to bindvarious DNA sequences. This archive was constructed by the Zinc FingerConsortium, a group of academic laboratories committed to thedevelopment of engineered zinc-finger technology (world-wide-web atzincfingers.org). They employed OPEN to rapidly and successfullyengineer a large set of zinc-finger arrays: 269 unique multi-fingerarrays for 34 different target sites. The inventors then used a subsetof these arrays to construct 37 active ZFN pairs for 11 different targetsites located within three endogenous human genes (VEGF-A, HoxB13,CFTR), an endogenous plant gene (tobacco SuRA), and the EGFP reportergene. Using these ZFNs, they show that: (i) OPEN is significantly morerobust than the existing modular assembly engineering method, (ii) OPENZFNs induce stable genome modifications with high efficiency (absoluterates ranging from 1-50% and with changes in as many as four alleles ina single cell), and (iii) OPEN ZFNs possess activities and toxicitiescomparable to those of an extensively optimized ZFN pair previouslyreported.

Moreover, the inventors have identified specific ZFNs that are designedto target the cystic fibrosis transmembrane conductance regulator (CFTR)gene, thereby providing a potential therapy for cystic fibrosis. In thepresent embodiment the focus is on modification of exon 10 of the CFTRgene, site of the most common human disease associated mutation, ΔF508,cause by a 3 bp deletion. Delivery methods and vectors for expressionZFNs are provided that enhance the homologous recombination within theCFTR gene. These and other aspects of the invention are describedfurther below.

I. ZINC FINGER NUCLEASES

A. Zinc Finger Proteins Zinc fingers are part of a large superfamily ofprotein domains that can bind to DNA. A zinc finger consists of twoantiparallel β strands, and an α helix. The zinc ion is crucial for thestability of this domain type—in the absence of the metal ion the domainunfolds as it is too small to have a hydrophobic core. One very wellexplored subset of zinc-fingers (the C₂H₂ class) comprises a pair ofcysteine residues in the beta strands and two histidine residues in thealpha helix which are responsible for binding a zinc ion. The two otherclasses of zinc finger proteins are the C₄ and C₆ classes. Zinc fingersare important in regulation because when interacted with DNA and zincion, they provide a unique structural motif for DNA-binding proteins.

The structure of each individual finger is highly conserved and consistsof about 30 amino acid residues, constructed as a ββα fold and heldtogether by the zinc ion. The α-helix occurs at the C-terminal part ofthe finger, while the β-sheet occurs at the N-terminal part. This ismost useful in transcription process. The consensus sequence of a singlefinger is:

Cys-X₂₋₄-Cys-X₃-Phe-X₅-Leu-X₂-His-X₃-His  (SEQ ID NO:113)

Many transcription factors (such as Zif268), regulatory proteins, andother proteins that interact with DNA contain zinc fingers. Theseproteins typically interact with the major groove along the double helixof DNA in which case the zinc fingers are arranged around the DNA strandin such a way that the α-helix of each finger contacts the DNA, formingan almost continuous stretch of α-helices around the DNA molecule. Someprimary neuron-specific transcriptional regulator that may be involvedin mediating early neural development are also zinc finger-based.

The binding specificity for 3-4 base pairs is conferred by a shortstretch of amino acid residues in the α-helix. The primary position ofthe amino acid residues within the α-helix interacting with the DNA areat positions −1, 3 and 6 relative to the first amino acid residue of theα-helix. Other amino acid positions can also influence bindingspecificity by assisting amino acid residues to bind a specific base orby contacting a fourth base in the opposite strand, causing target-siteoverlap.

B. ZFNs

Zinc finger nucleases (ZFNs) are protein chimera comprised of a zincfinger-based DNA-binding domain and a DNA-cleavage domain. They are ableto introduce double-strand breaks (DSB; breaks at the same or very closepoints in both strands of a double-stranded DNA molecule) at specificlocations within a DNA molecule which may subsequently be used todisable a specific allele or even rewrite the code it contains. Inventorof the zinc finger nuclease is Srinivasan Chandrasegaran from JohnsHopkins University in Baltimore, Md. ZFNs are undergoing development foruse in gene therapy and research applications.

The DNA-binding domain of a ZFN may be composed of two to six zincfingers due to their supposed modularity (appositeness to be usedinterchangeably). Each zinc finger motif is typically considered torecognise and bind to a three-base pair sequence and as such, a proteinincluding more zinc fingers targets a longer sequence and therefore hasa greater specificity and affinity to the target site. Depending uponthe required specifications of the end-product, the included zincfingers may be selected via a parallel, sequential or bipartitetechnique or through an in vitro cell-based technique.

The non-specific nuclease domain of FokI is functionally independent ofits natural DNA-binding domain and is therefore employed in theconstruction of ZFNs. Since the domain must dimerise to accomplish adouble-strand break it is necessary that a nuclease is also bound to theopposite strand by virtue of another ZFN molecule bound to its targetsequence as shown in the diagram. The two target sites need not be thesame, so long as ZFNs targeting both sites are present. In order to forma dimer, two ZFN molecules must meet with their respective recognitionsites not less than 4-6 base pairs apart but also not so far apart thatthey may not dimerise. While one ZFN molecule binds its target sequenceon one strand, another ZFN molecule binds its target sequence on theopposite strand, as shown in the diagram. The nuclease domains dimeriseand each cleaves its own strand, producing a DSB. FokI can be employedas a homo- or a heterodimer. The advantage of the heterodimer is that itmay reduce off target effects (Miller et al., 2007).

ZFNs can be used to disable dominant mutations in heterozygousindividuals by producing DSBs in the mutant allele which will, in theabsence of a homologous template, be repaired by non-homologousend-joining (NHEJ). NHEJ repairs double-strand breaks by joining the twoends together and usually produces no mutations, provided that the cutis clean and uncomplicated. In some instances however, the repair willbe imperfect, resulting in deletion or insertion of base-pairs,producing frame-shift and preventing the production of the harmfulprotein.

C. Expression Systems for ZFNs

In particular embodiments, the ZFN may advantageously be delivered to ahost cell or subject using a recombinant vector encoding the ZFN. Theterm “recombinant” generally refers to a polypeptide produced from anucleic acid molecule that has been manipulated in vitro or that is thereplicated product of such a molecule.

The nucleic acid segments used in the present invention, regardless ofthe length of the coding sequence itself, may be combined with othernucleic acid sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (Sambrook et al., 1989; Ausubel et al., 1996, bothincorporated herein by reference). In addition to encoding a modifiedpolypeptide such as modified gelonin, a vector may encode non-modifiedpolypeptide sequences such as a tag or targetting molecule. Usefulvectors encoding such fusion proteins include pIN vectors (Inouye etal., 1985), vectors encoding a stretch of histidines, and pGEX vectors,for use in generating glutathione S-transferase (GST) soluble fusionproteins for later purification and separation or cleavage. A targettingmolecule is one that directs the modified polypeptide to a particularorgan, tissue, cell, or other location in a subject's body.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

1. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind such as RNA polymerase and other transcriptionfactors. The phrases “operatively positioned,” “operatively linked,”“under control,” and “under transcriptional control” mean that apromoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence. A promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other prokaryotic, viral, or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. In addition to producing nucleicacid sequences of promoters and enhancers synthetically, sequences maybe produced using recombinant cloning and/or nucleic acid amplificationtechnology, including PCR™, in connection with the compositionsdisclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906,each incorporated herein by reference). Furthermore, it is contemplatedthe control sequences that direct transcription and/or expression ofsequences within non-nuclear organelles such as mitochondria,chloroplasts, and the like, can be employed as well.

Naturally, it may be important to employ a promoter and/or enhancer thateffectively directs the expression of the DNA segment in the cell type,organelle, and organism chosen for expression. Those of skill in the artof molecular biology generally know the use of promoters, enhancers, andcell type combinations for protein expression, for example, see Sambrooket al. (1989), incorporated herein by reference. The promoters employedmay be constitutive, tissue-specific, inducible, and/or useful under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The promoter may be heterologousor endogenous. The identity of tissue-specific promoters or elements, aswell as assays to characterize their activity, is well known to those ofskill in the art.

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′-methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, herein incorporated by reference). Thepicornavirus-derived, self-cleaving 2A peptide, designated T2A, allowsfor the efficient translation of multiple cistrons and therefore is anadditional element that can be employed in multi-cistronic messages.

3. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. (See Carbonelli et al., 1999, Levenson et al., 1998,and Cocea, 1997, incorporated herein by reference.) “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is widely understood by those of skill in the art.Frequently, a vector is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the vector. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology.

4. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression. (SeeChandler et al., 1997, incorporated herein by reference.)

5. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and/or to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

6. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and/or any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal and/or the bovine growth hormone polyadenylationsignal, convenient and/or known to function well in various targetcells. Polyadenylation may increase the stability of the transcript ormay facilitate cytoplasmic transport.

7. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

8. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

9. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organisms that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid, such as a modified protein-encoding sequence, istransferred or introduced into the host cell. A transformed cellincludes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, includingyeast cells, insect cells, and mammalian cells, depending upon whetherthe desired result is replication of the vector or expression of part orall of the vector-encoded nucleic acid sequences. Numerous cell linesand cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials (world-wide-web at atcc.org). An appropriate host canbe determined by one of skill in the art based on the vector backboneand the desired result. A plasmid or cosmid, for example, can beintroduced into a prokaryote host cell for replication of many vectors.Bacterial cells used as host cells for vector replication and/orexpression include DH5α, JM109, and KC8, as well as a number ofcommercially available bacterial hosts such as SURE® Competent Cells andSOLOPACK™ Gold Cells (STRATAGENE®, La Jolla, Calif.). Alternatively,bacterial cells such as E. coli LE392 could be used as host cells forphage viruses. Appropriate yeast cells include Saccharomyces cerevisiae,Saccharomyces pombe, and Pichia pastoris.

Examples of eukaryotic host cells for replication and/or expression of avector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Manyhost cells from various cell types and organisms are available and wouldbe known to one of skill in the art. Similarly, a viral vector may beused in conjunction with either a eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

10. Commercial Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

In addition to the disclosed expression systems of the invention, otherexamples of expression systems include STRATAGENE®'s COMPLETE CONTROL™Inducible Mammalian Expression System, which involves a syntheticecdysone-inducible receptor, or its pET Expression System, an E. coliexpression system. Another example of an inducible expression system isavailable from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

11. Viral Vectors

There are a number of ways in which expression vectors may be introducedinto cells. In certain embodiments of the invention, the expressionvector comprises a virus or engineered vector derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubinstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kb of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubinstein, 1988; Temin, 1986).

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells; they can also be used as vectors. Lentiviruses arecomplex retroviruses, which, in addition to the common retroviral genesgag, pol, and env, contain other genes with regulatory or structuralfunction. Lentiviral vectors are well known in the art (see, forexample, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al.,1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 andthe Simian Immunodeficiency Virus: SIV. Lentiviral vectors have beengenerated by multiply attenuating the HIV virulence genes, for example,the genes env, vif, vpr, vpu and nef are deleted making the vectorbiologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat is describedin U.S. Pat. No. 5,994,136, incorporated herein by reference. One maytarget the recombinant virus by linkage of the envelope protein with anantibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

12. Methods of Gene Transfer

Suitable methods for nucleic acid delivery to effect expression ofcompositions of the present invention are believed to include virtuallyany method by which a nucleic acid (e.g., DNA, including viral andnonviral vectors) can be introduced into an organelle, a cell, a tissueor an organism, as described herein or as would be known to one ofordinary skill in the art. Such methods include, but are not limited to,direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harland and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference); by calciumphosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama,1987; Rippe et al., 1990); by using DEAE-dextran followed bypolyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimeret al., 1987); by liposome mediated transfection (Nicolau and Sene,1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980;Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment(PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos.5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, andeach incorporated herein by reference); by agitation with siliconcarbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and5,464,765, each incorporated herein by reference); byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); or by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985).Through the application of techniques such as these, organelle(s),cell(s), tissue(s) or organism(s) may be stably or transientlytransformed.

II. CYSTIC FIBROSIS AND CFTR

A. Cystic Fibrosis (CF)

Cystic Fibrosis (also known as CF) is a hereditary disease affecting theexocrine (mucus) glands of the lungs, liver, pancreas, and intestines,causing progressive disability due to multisystem failure. Abnormallythick mucus results in frequent lung infections. Diminished secretion ofpancreatic enzymes is the main cause of poor growth, greasy stools, anddeficiency in fat-soluble vitamins. Males can be infertile due to thecondition congenital bilateral absence of the vas deferens. Often,symptoms of CF appear in infancy and childhood. Meconium ileus is atypical finding in newborn babies with CF.

There is no known cure for CF, and most individuals with cystic fibrosisdie young: many in their 20's and 30's from lung failure. The predictedmedian age of survival for a person with CF is 37 years. However, withthe continuous introduction of many new treatments, the life expectancyof a person with CF is increasing to ages as high as 40 or 50. Lungtransplantation is often necessary as CF worsens.

Cystic fibrosis is one of the most common life-shortening geneticdiseases. In the United States, 1 in 4,000 children is born with CF. Itis most common among western European populations; one in twenty-twopeople of Mediterranean descent is a carrier of one gene for CF, makingit the most common genetic disease in these populations. In 1997, about1 in 3,300 caucasian children in the United States was born with cysticfibrosis. In contrast, only 1 in 15,000 African American childrensuffered from cystic fibrosis, and in Asian Americans the rate was evenlower at 1 in 32,000.

CF is caused by a mutation in the gene, cystic fibrosis transmembraneconductance regulator (CFTR). The product of this gene is a chloride ionchannel important in creating sweat, digestive juices, and mucus.Although most people without CF have two working copies (alleles) of theCFTR gene, only one is needed to prevent cystic fibrosis. CF developswhen neither allele can produce a functional CFTR protein. Therefore, CFis considered an autosomal recessive disease.

Cystic fibrosis may be diagnosed by many different categories of testingincluding those such as, newborn screening, sweat testing, or genetictesting. As of 2006 in the United States, 10 percent of cases arediagnosed shortly after birth as part of newborn screening programs. Thenewborn screen initially measures for raised blood concentration ofimmunoreactive trypsinogen. Infants with an abnormal newborn screen needa sweat test in order to confirm the CF diagnosis. Trypsinogen levelscan be increased in individuals who have a single mutated copy of theCFTR gene (carriers) or, in rare instances, even in individuals with twonormal copies of the CFTR gene. Due to these false positives, CFscreening in newborns is somewhat controversial. Most states andcountries do not screen for CF routinely at birth. Therefore, mostindividuals are diagnosed after symptoms prompt an evaluation for cysticfibrosis. The most commonly-used form of testing is the sweat test.Sweat-testing involves application of a medication that stimulatessweating (pilocarpine) to one electrode of an apparatus and runningelectric current to a separate electrode on the skin. This process,called iontophoresis, causes sweating; the sweat is then collected onfilter paper or in a capillary tube and analyzed for abnormal amounts ofsodium and chloride. People with CF have increased amounts of sodium andchloride in their sweat. CF can also be diagnosed by identification ofmutations in the CFTR gene.

A multitude of tests are used to identify complications of CF and tomonitor disease progression. X-rays and CAT scans are used to examinethe lungs for signs of damage or infection. Examination of the sputumunder a microscope is used to identify which bacteria are causinginfection so that effective antibiotics can be given. Pulmonary functiontests measure how well the lungs are functioning, and are used tomeasure the need for and response to antibiotic therapy. Blood tests canidentify liver abnormalities, vitamin deficiencies, and the onset ofdiabetes. DEXA scans can screen for osteoporosis and testing for fecalelastase can help diagnose insufficient digestive enzymes.

As discussed above, cystic fibrosis occurs when there is a mutation inboth copies of the CFTR gene. The protein created by this gene isanchored to the outer membrane of cells in the sweat glands, lungs,pancreas, and other affected organs. The protein spans this membrane andacts as a channel connecting the inner part of the cell (cytoplasm) tothe surrounding fluid. In the airway this channel is primarilyresponsible for controlling the movement of chloride from inside tooutside of the cell, however in the sweat ducts it facilitates themovement of chloride from the sweat into the cytoplasm. When the CFTRprotein does not work, chloride is trapped inside the cells in theairway and outside in the skin. Because chloride is negatively charged,positively charged ions cross into the cell because they are affected bythe electrical attraction of the chloride ions. Sodium is the mostcommon ion in the extracellular space and the combination of sodium andchloride creates the salt, which is lost in high amounts in the sweat ofindividuals with CF. This lost salt forms the basis for the sweat test.

How this malfunction of cells in cystic fibrosis causes the clinicalmanifestations of CF is not well understood. One theory suggests thatthe lack of chloride exodus through the CFTR protein leads to theaccumulation of more viscous, nutrient-rich mucus in the lungs thatallows bacteria to hide from the body's immune system. Another theoryproposes that the CFTR protein failure leads to a paradoxical increasein sodium and chloride uptake, which, by leading to increased waterreabsorption, creates dehydrated and thick mucus. Yet another theoryfocuses on abnormal chloride movement out of the cell, which also leadsto dehydration of mucus, pancreatic secretions, biliary secretions, etc.These theories all support the observation that the majority of thedamage in CF is due to blockage of the narrow passages of affectedorgans with thickened secretions. These blockages lead to remodeling andinfection in the lung, damage by accumulated digestive enzymes in thepancreas, blockage of the intestines by thick faeces, etc.

B. CFTR

The CFTR gene is found at the q31.2 locus of chromosome 7, is 230,000base pairs long, and creates a protein that is 1,480 amino acids long.The most common mutation, ΔF508 is a deletion (Δ) of three nucleotidesin exon 10 that results in a loss of the amino acid phenylalanine (F) atthe 508th (508) position on the protein. This mutation accounts fortwo-thirds of CF cases worldwide and 90% of cases in the United States;however, there are over 1,400 other mutations that can produce CF.

There are several mechanisms by which these mutations cause problemswith the CFTR protein. ΔF508, for instance, creates a protein that doesnot fold normally and is degraded by the cell. Several mutations, whichare common in the Ashkenazi Jewish population, result in proteins thatare too short because production is ended prematurely. Less commonmutations produce proteins that do not use energy normally, do not allowchloride to cross the membrane appropriately, or are degraded at afaster rate than normal. Mutations may also lead to fewer copies of theCFTR protein being produced.

Structurally, CFTR is a type of gene known as an ABC gene. Its proteinpossesses two ATP-hydrolyzing domains which allows the protein to useenergy in the form of ATP. It also contains two domains comprising 6alpha helices apiece, which allow the protein to cross the cellmembrane. A regulatory binding site on the protein allows activation byphosphorylation, mainly by cAMP-dependent protein kinase. The carboxylterminal of the protein is anchored to the cytoskeleton by a PDZ domaininteraction.

Reference to the human CFTR sequence is made by way of Genbank AccessionNo. NM_(—)000492 (SEQ ID NO:114 and 115) as well as SEQ ID NOS: 1-2.

III. TREATMENT OF CF WITH ZFNS

A. ZFNs Targeting Δ508F

In one embodiment, the present invention provides for the treatment ofCF using particular ZFNs identified herein. These ZFNs target a regionof the CFTR that is adjacent to (w/i 100 base of) the Δ508F mutation.Reference to exon 10 of the CFTR sequence is made in FIG. 10.

B. Pharmaceutical Compositions and Routes of Administration

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more ZFNs dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one ZFN, and optionally an additional activeingredient, will be known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company (1990), incorporated herein byreference. Moreover, for animal (e.g., human) administration, it will beunderstood that preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The ZFNs may be admixed with different types of carriers depending onhow they are be administered. The present invention can be administeredbuccally, intravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, topically, intramuscularly, intratumorally, into tumorvasculature, subcutaneously, mucosally, orally, topically, locally,inhalation (e.g., aerosol inhalation), injection, infusion, continuousinfusion, localized perfusion bathing target cells directly, via acatheter, via a lavage, in cremes, in lipid compositions (e.g.,nanoparticles, liposomes), or by other method or any combination of theforgoing as would be known to one of ordinary skill in the art (see, forexample, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990, incorporated herein by reference). In particular, theZFNs is formulated into a syringeable composition for use in intravenousadministration.

The ZFNs may be formulated into a composition in a free base, neutral orsalt form or ester. Pharmaceutically acceptable salts, include the acidaddition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric, fumaric, or mandelic acid. Salts formedwith the free carboxyl groups can also be derived from inorganic basessuch as for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent. Acarrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of the composition contained therein, its usein administrable composition for use in practicing the methods of thepresent invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include ZFNs, one or morelipids, and an aqueous solvent. As used herein, the term “lipid” will bedefined to include any of a broad range of substances that ischaracteristically insoluble in water and extractable with an organicsolvent. This broad class of compounds are well known to those of skillin the art, and as the term “lipid” is used herein, it is not limited toany particular structure. Examples include compounds which containlong-chain aliphatic hydrocarbons and their derivatives. A lipid may benaturally-occurring or synthetic (i.e., designed or produced by man).Lipids are well known in the art, and include for example, neutral fats,phospholipids, phosphoglycerides, steroids, terpenes, lysolipids,glycosphingolipids, glycolipids, sulphatides, lipids with ether andester-linked fatty acids and polymerizable lipids, and combinationsthereof.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the ZFNs may be dispersed in a solution containinga lipid, dissolved with a lipid, emulsified with a lipid, mixed with alipid, combined with a lipid, covalently bonded to a lipid, contained asa suspension in a lipid, contained or complexed with a micelle orliposome, or otherwise associated with a lipid or lipid structure by anymeans known to those of ordinary skill in the art. The dispersion may ormay not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, ZFNs pharmaceutical compositions may comprise,for example, at least about 0.1% of the ZFN, about 0.5% of the ZFN, orabout 1.0% of the ZFN. In other embodiments, the ZFN may comprisebetween about 2% to about 75% of the weight of the unit, or betweenabout 25% to about 60%, for example, and any range derivable therein.Naturally, the amount of the ZFN in each therapeutically usefulcomposition may be prepared is such a way that a suitable dosage will beobtained in any given unit dose of the compound. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

In other non-limiting examples, a dose of a ZFN may also comprise fromabout 0.1 microgram/kg/body weight, about 0.2 microgram/kg/body weight,about 0.5 microgram/kg/body weight, about 1 microgram/kg/body weight,about 5 microgram/kg/body weight, about 10 microgram/kg/body weight,about 50 microgram/kg/body weight, about 100 microgram/kg/body weight,about 200 microgram/kg/body weight, about 350 microgram/kg/body weight,about 500 microgram/kg/body weight, about 1 milligram/kg/body weight,about 5 milligram/kg/body weight, about 10 milligram/kg/body weight,about 50 milligram/kg/body weight, about 100 milligram/kg/body weight,about 200 milligram/kg/body weight, about 350 milligram/kg/body weight,about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight ormore per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In particular embodiments of the present invention, the ZFNs areformulated to be administered via an alimentary route. Alimentary routesinclude all possible routes of administration in which the compositionis in direct contact with the alimentary tract. Specifically, thepharmaceutical compositions disclosed herein may be administered orally,buccally, rectally, or sublingually. As such, these compositions may beformulated with an inert diluent or with an assimilable edible carrier,or they may be enclosed in hard- or soft-shell gelatin capsule, or theymay be compressed into tablets, or they may be incorporated directlywith the food of the diet.

For oral administration, the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, gel or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet, gel or solution form that may be placed under the tongue, alongthe gum line, brushed on to teeth surfaces, or otherwise dissolved inthe mouth. U.S. Pat. Nos. 6,074,674 and 6,270,750, both incorporated byreference, describe topical, sustained release compositions forperiodontal procedures.

In further embodiments, ZFNs may be administered via a parenteral route.As used herein, the term “parenteral” includes routes that bypass thealimentary tract. Specifically, the pharmaceutical compositionsdisclosed herein may be administered for example, but not limited tointravenously, intradermally, intramuscularly, intraarterially,intrathecally, subcutaneous, or intraperitoneally. Solutions of theactive compounds as free base or pharmacologically acceptable salts maybe prepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions may also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms. The pharmaceuticalforms suitable for injectable use include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468,specifically incorporated herein by reference in its entirety). In allcases the form must be sterile and must be fluid to the extent that easyinjectability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (i.e., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. The prevention ofthe action of microorganisms can be brought about by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

Of particular relevance to the present invention is aerosoladministration, also known as inhalation therapy, is the administrationof ZFNs by an appropriate device that permits inhalation and absorptioninto the patient's lungs. Aerosol administration in itself is generallya safe practice, as long as the health care provider or user is welleducated in its use. It is contraindicated in conditions where completeobstruction of the airway is present, as the administration route iscompletely blocked. Aerosol drug administration, or in some casesnebulized drug therapy, disperses drugs into the lungs or bronchialairways in the form of tiny droplets—often bound to water, oxygen, oranother gaseous substance. Drugs are generally delivered by two means.The first is via a device called a nebulizer. The nebulizer is amechanical pump (of which there are many types) that produces a finemist in which the drug is dispersed via an appropriatenebulizer-compatible face mask. This fine mist is inhaled deep into thelungs for maximum effect. The second method of delivery is via ahand-held, nebulized aerosol device. These devices, also known as“puffers,” use the effects of a pressurized gas to create and dispersethe drug into a fine mist or spray, which is then inhaled. Both methodsof aerosol inhalation are very effective when used correctly.

C. Combination Therapies

In some aspects of the present invention, other agents may be used incombination with ZFNs to provide a more effective therapy for CF. Moregenerally, these agents would be provided in a combined amount toproduce or increase any of the effects discussed herein. This processmay involve contacting a subject with both agents at the same time. Thismay be achieved by contacting the cell or subject with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell or subject with two distinct compositions orformulations, at the same time, wherein one composition includes theintracellular Cathepsin L inhibitor and the other includes the secondagent.

Alternatively, one agent may precede or follow the other by intervalsranging from minutes to weeks. In embodiments where the agents areapplied separately to the cell or subject, one would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the agents would still be able to exert anadvantageously combined effect on the cell or subject. In suchinstances, it is contemplated that one may contact the cell with bothmodalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

Various combinations may be employed, the ZFN is “A” and the other agentis “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/AAdministration protocols and formulation of such agents will generallyfollow those of standard pharmaceutical drugs, as discussed furtherbelow.

Many CF patients are on one or more antibiotics at all times, even whenthey are considered healthy, to suppress the infection as much aspossible. Antibiotics are absolutely necessary whenever pneumonia issuspected or there has been a noticeable decline in lung function.Antibiotics are usually chosen based on the results of a sputum analysisand the patient's past response. Many bacteria common in cystic fibrosisare resistant to multiple antibiotics and require weeks of treatmentwith intravenous antibiotics such as vancomycin, tobramycin, meropenem,ciprofloxacin, and piperacillin. This prolonged therapy oftennecessitates hospitalization and insertion of a more permanent IV suchas a PICC line or Port-a-Cath. Inhaled therapy with antibiotics such astobramycin and colistin is often given for months at a time in order toimprove lung function by impeding the growth of colonized bacteria.Inhaled therapy with the antibiotic aztreonam is also being developedand clinical trials have shown great promise. Oral antibiotics such asciprofloxacin or azithromycin are given to help prevent infection or tocontrol ongoing infection. Some individuals spend years betweenhospitalizations for antibiotics, whereas others require severalantibiotic treatments each year.

Several common antibiotics such as tobramycin and vancomycin can causehearing loss, damage to the balance system in the inner ear or kidneyproblems with long-term use. In order to prevent these side-effects, theamount of antibiotics in the blood are routinely measured and adjustedaccordingly.

Several mechanical techniques are used to dislodge sputum and encourageits expectoration. In the hospital setting, physical therapy isutilized; a therapist percusses an individual's chest with his or herhands several times a day. Devices that recreate this percussive therapyinclude the ThAIRapy Vest and the intrapulmonary percussive ventilator(IPV). Newer methods such as Biphasic Cuirass Ventilation, andassociated clearance mode available in such devices, now integrate acough assistance phase, as well as a vibration phase for dislodgingsecretions. Biphasic Cuirass Ventilation is also shown to provide abridge to transplantation. These are portable and adapted for home use.Physiotherapy is essential to help manage an individuals chest on a longterm basis, and can also teach techniques for the older child andteenager to manage themselves at home. Aerobic exercise is of greatbenefit to people with cystic fibrosis. Not only does exercise increasesputum clearance but it also improves cardiovascular and overall health.

Aerosolized medications that help loosen secretions include dornase alfaand hypertonic saline. Dornase is a recombinant human deoxyribonuclease,which breaks down DNA in the sputum, thus decreasing its viscosity.N-Acetylcysteine may also decrease sputum viscosity, but research andexperience have shown its benefits to be minimal. Albuterol andipratropium bromide are inhaled to increase the size of the smallairways by relaxing the surrounding muscles.

As lung disease worsens, breathing support from machines may becomenecessary. Individuals with CF may need to wear special masks at nightthat help push air into their lungs. These machines, known as bilevelpositive airway pressure (BiPAP) ventilators, help prevent low bloodoxygen levels during sleep. BiPAP may also be used during physicaltherapy to improve sputum clearance. During severe illness, people withCF may need to have a tube placed in their throats (a procedure known asa tracheostomy) and their breathing supported by a ventilator.

Lung transplantation often becomes necessary for individuals with cysticfibrosis as lung function and exercise tolerance declines. Althoughsingle lung transplantation is possible in other diseases, individualswith CF must have both lungs replaced because the remaining lung wouldcontain bacteria that could infect the transplanted lung. A pancreaticor liver transplant may be performed at the same time in order toalleviate liver disease and/or diabetes. Lung transplantation isconsidered when lung function approaches a point where it threatenssurvival or requires assistance from mechanical devices. This point istypically when lung function declines to approximately 20 to 30 percent,however the there is a small time frame when transplantation is feasibleas the patient must be healthy enough to endure the procedure.

IV. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials & Methods

Construction of Zinc-Finger Pools.

A detailed description of how OPEN zinc-finger pools were constructed isprovided in Supplementary Experimental Procedures. In brief, zinc-fingerlibraries were constructed using cassette mutagenesis to randomize therecognition helix of one finger in a threefinger domain. “B2H selectionstrains” harboring the target nine by site were constructed as described(Thibodeau-Beganny and Joung, 2007). Zinc-finger pools were obtainedusing two selection steps: First, randomized library members wereintroduced into a B2H selection strain and plated on histidine-deficientselective media (NM media) containing 3-AT, a competitive inhibitor ofthe HIS3 enzyme. Surviving colonies were scraped and infected withM13K07 helper phage to rescue zinc-finger-encoding phagemids asinfectious phage particles. Second, rescued phagemids were used toinfect fresh B2H selection cells and plated on NM media containing 3-ATand streptomycin. 95 surviving colonies were picked and inoculated intoa 96-well block for growth and plasmid DNA isolated to obtain the finalfinger pools (FIG. 1A).

OPEN Selections.

A detailed description of how OPEN selections were performed is providedin Supplementary Experimental Procedures. In brief, for each targetsite, PCR-mediated fusion was used to create phage-based librariesconsisting of random combinations of three finger pools (FIG. 1B)expressed as fusions to a fragment of the Gal11P protein (FIG. 1C). OPENselections were performed in two steps. First, B2H selection straincells were infected with randomized zinc-finger phage library and thenplated on NM media containing 3-AT and streptomycin. Surviving colonieswere scraped and infected with M13K07 helper phage to rescue thezinc-finger-encoding phagemids as phage. In a second step, this enrichedphage library was then used to re-infect fresh B2H selection straincells which were then plated on NM media containing a gradient of 3-ATand streptomycin. For a small number of the OPEN selections, theinventors performed selections in a single step (see below).

Quantitative Bacterial Two-Hybrid (B2H) Assays.

Zinc-finger-encoding plasmids identified from OPEN selections wereco-transformed with an α-Gal4 expression plasmid into a “B2H reporterstrain” harboring a single copy bacterial plasmid with a target bindingsite positioned upstream of a weak promoter driving lacZ expression. B2Hreporter strains were constructed as described (Wright et al., 2006).β-galactosidase assays were performed in triplicate as described(Thibodeau et al., 2004).

Human cell-based EGFP-disruption assay. Human 293.EGFP cells harbor anintegrated retroviral construct which constitutively expresses aβ-galactosidase-EGFP fusion protein (FIG. 2C). 50,000 transfected cellswere analyzed by flow cytometry two and five days post-transfection todetermine the percentage of EGFP-negative cells. Statisticalsignificance was determined using a two-sided student's t-test withunequal variance.

CEL I Nuclease Assay for NHEJ-Mediated Mutation.

Flp-In T-REx 293 cells (Invitrogen) were transfected with ZFN expressionplasmids and genomic DNA was isolated three days post-transfection.Limited-cycle PCR was performed with radiolabeled nucleotides andVEGF-A- or HoxB13-specific primers. PCR products were treated with CEL Inuclease and then separated on 10% polyacrylamide gels and visualizedusing a phosphorimaging screen. All experiments were performed a minimumof two times.

Gene Targeting Assays.

Human Flp-In T-REx 293 and K562 cells were transfected with ZFNexpression plasmids and donor constructs and genomic DNA harvest threeand four days post-transfection. Limited-cycle PCR was performed withradiolabeled nucleotides and VEGF-A- or IL2Rγ-specific primers. PCRproducts digested with SalI (for VEGF-A) or BsrBI (for IL2Rγ) wereseparated on 10% polyacrylamide gels and visualized using aphosphorimaging screen. Additional details and Southern blot assays aredescribed below.

Tobacco Transformation and Assay for Mutations.

The transformation of tobacco protoplasts by electroporation was carriedout as previously described (Wright et al., 2005). Protoplasts wereallowed to recover and then selected for kanamycin resistance andregenerated into plantlets as previously described (Wright et al.,2005). DNA was prepared from tissue harvested from individual plantletsusing the Epicentre MasterPure Plant leaf DNA Purification Kit. SuRA andSuRB alleles were amplified by PCR, gel purified, and sequenced toidentify mutations (see Supplementary Experimental Procedures foradditional details).

Sequencing of Modified Genomic Alleles.

PCR products amplified from genomic DNA were cloned into thepCR4Blunt-TOPO plasmid using the Zero Blunt TOPO PCR Cloning Kit forSequencing (Invitrogen). Plasmid DNA was isolated from transformants andsequenced using primers as described in Supplementary ExperimentalProcedures.

ZFN Toxicity Assays.

ZFN expression vectors, donor templates, and plasmid pmaxGFP (Amaxa)were transfected into K562 cells. Cells were assayed for GFP expressionat days 1 and 7 posttransfection using a FACScan cytometer and for genetargeting efficiencies at days 4 and 7 post-transfection using thelimited-cycle PCR/restriction digest assay. Variant heterodimer FokIdomains were constructed by introducing the “+” and “−” mutationspreviously described in Miller et al. (2007).

Fluorescence In Situ Hybridization (FISH).

Two-color fluorescence in situ hybridization (FISH) was performed on 3:1methanolacetic acid fixed cell lines using bacterial artificialchromosome clones RP11-710L16 (6p21.1; VEGFA) labeled in Spectrum Orange(Abbott-Vysis, Downer's Grove, Il.), or RP11-142P4 (14q31.1; copy numbercontrol) labeled in Spectrum Green using standard protocols. Images werecaptured using an Olympus BX61 fluorescent microscope equipped with aCCD camera, and analysis was performed with Cytovision software (AppliedImaging, San Jose, Calif.).

B2H Selection Media.

NM medium has been previously described (Thibodeau-Beganny and Joung,2007). NM/CCK medium plates contain 100 μg/mL carbenicillin, 30 μg/mLchloramphenicol, 30 μg/mL kanamycin, and 1.5% Bacto-agar.

Construction of Zinc Finger Pools.

All master randomized zinc finger libraries were constructed in astandard framework consisting of three tandem repeats of the middlefinger of the murine transcription factor Zif268 in which therecognition helix residues have been altered. For each library,recognition helix residues −1, 1, 2, 3, 5, and 6 were randomized using24 codons (degenerate sequence 5′VNS3′) encoding 16 amino acids(excluding cysteine and the aromatics). The theoretical complexity ofeach library is therefore 24⁶=˜2×10⁸ members.

B2H selection strains each harbor: (1) a single copy episome bearing atarget site of interest positioned upstream of a promoter which drivesco-cistronic expression of two selectable markers (the yeast HIS3 geneand the bacterial aadA gene) and (2) a low copy number plasmidexpressing the RNA polymerase α-subunit/yeast Ga14 hybrid protein(α-Ga14). Strains were constructed as previously described(Thibodeau-Beganny and Joung, 2007). The target binding site of eachstrain was verified by DNA sequencing.

Zinc finger pools were obtained using two selection steps. In a firststep, 10⁹ ampicillin-transducing units (ATU) of randomized zinc fingerphage library were introduced into >3×10⁹ B2H selection strain cellsharboring a target subsite of interest. Transformed cells were plated onhistidine-deficient NM/CCK medium plates containing 50 μM isopropylβ-d-thiogalactoside (IPTG) and 10 mM 3-aminotriazole (3-AT), acompetitive inhibitor of the HIS3 enzyme. After incubation for 24 hoursat 37° C. followed by 18 hours at room temperature, surviving colonieswere scraped from the plates and infected with M13K07 helper phage torescue the zinc finger-encoding phagemids as infectious phage. In asecond step, this enriched phage library was then used to re-infectfresh B2H selection strain cells and the resulting transformants platedon NM/CCK medium plates containing 50 μM IPTG, 10 mM 3-AT, and 20 μg/mlstreptomycin. After incubation at 37° C. for 48 hours, the inventorsinoculated 95 surviving colonies into individual wells of a 96-wellblock containing 1 ml of Terrific Broth and 50 μg/ml carbenicillin.These cultures were grown overnight at 37° C. and then a 96-pinreplicator was used to inoculate a second block of identical cultures.Glycerol was added to a final concentration of 15% to all wells in thefirst block and this was stored at −80° C. The second block was grownovernight at 37° C. and then the 95 cultures were pooled together.Plasmid DNA encoding the finger pools was isolated from 10 ml of thepooled culture using a QIAgen miniprep kit and for most pools a smallnumber of random clones were sequenced with primer OK61. For fingerpools against nine subsites, sequencing revealed no strong consensus(i.e., the sequences were diverse and did not resemble one another),suggesting that selective pressure for those sites was relatively weakunder the initial selection conditions. For these nine subsites, thesecond step of selections were repeated and plated on higher stringencyplates to obtain sequences that more closely resembled one another. Thehigher stringency plates used were NM/CCK medium plates containing 50 μMIPTG, 20 mM 3-AT, and 30 μg/ml streptomycin (for seven subsites: F1 GAT,F2 GAC, F2 GAG, F2 GCG, F2 TGA, F2 TAG, and F2 GTT) or 50 μM IPTG, 25 mM3-AT, and 40 μg/ml streptomycin (for two subsites: F2 GAA, F2 TGG).

OPEN Selections.

To create libraries for use in OPEN selections, finger pools wereamplified by PCR (FIG. 6). For each amplification, first five and then20 cycles of PCR were performed using the following primers andannealing temperatures (primer names; initial annealing temp; finalannealing temp): for finger 1: OK1424 and OK1425; 55° C.; 59° C., forfinger 2: OK1426 and OK1427; 52° C.; 57° C., for finger 3: OK1428 andOK1429; 41° C.; 56° C. Amplification of individual finger pools wereisolated from 10% polyacrylamide gels and fused together by PCR. To dothis, equal concentrations of the three finger pool fragments were fusedusing the following PCR conditions: 94° C., 5 minutes; 10 cycles of 94°C., 30 sec; 50° C., 30 sec; 72° C., 2 min; final extension 72° C., 7min. This fusion product was purified using a QIAgen PCR purificationkit and then amplified by PCR using primers OK1430 and OK1432 with 10initial cycles of 94° C., 30 sec; 56° C., 30 sec; 72° C., 1 min and 20additional cycles of 94° C., 30 sec; 64° C., 30 sec; 72° C., 1 min;final extension 72° C., 7 min. The final PCR product (encoding a libraryof three-finger arrays) was isolated on a 5% polyacrylamide gel andtreated with Pfu polymerase and T4 polynucleotide kinase to createoverhangs. This fragment was then ligated with BbsI-digestedpBR-UV5-GP-FD2 vector backbone which results in a plasmid that expressesthe zinc finger array as a FLAG-tagged Gal11P fusion in the B2H system.This ligation was then introduced into E. coli XL-1 Blue cells byelectroporation and each library was constructed from >3×10₆ independenttransformants, ensuring at least three-fold oversampling of thetheoretical library complexity of ˜8.6×10⁵ (95³).

Libraries were then converted into infectious M13 phage as previouslydescribed (Thibodeau-Beganny and Joung, 2007). All but six (see below)of the OPEN selections were performed in two stages. In the first stage,an OPEN three-finger library was introduced by infection into a B2Hselection strain harboring the full target DNA sequence of interest.>2.2×10⁶ ATU of OPEN phage library were used to infect >2×10⁸ B2Hselection strain cells and the resulting transformants were plated ontwo different NM/CCK medium plates containing 50 μM IPTG, 10 mM 3AT, and20 μg/mL streptomycin or 50 μM IPTG, 25 mM 3AT, and 40 μg/mLstreptomycin. After 36-48 hours of incubation, colonies were harvestedfrom the highest stringency plate yielding at least 1000 colonies. Thesecells were infected with M13K07 helper phage to rescue zincfinger-encoding phagemids, thereby creating an enriched library. In thesecond stage, 2.5×10⁶ ATU of this enriched library were used to infect>2×10⁸ fresh B2H selection strain cells and the resulting transformantsplated on a 245×245 mm NM/CCK medium plate containing parallel gradientsof 3-AT and streptomycin ranging from 0 mM to 80 mM and 0 μg/mL to 100μg/mL, respectively.

Gradient plates were poured using the method of Szybalski (Bryson andSzybalski, 1952). After incubation at 37° C. for a minimum of 48 and amaximum of 96 hours, 12 surviving colonies were picked from the higheststringency edge of the plate and ZFP-encoding plasmids were isolated byplasmid miniprep and sequenced with primer OK61. For six of the sites,the inventors targeted using OPEN, selections were performed in a singlestep (instead of two steps). For five sites (EG223L, EG223R, EG292L,EG292R, and EG382L), >3.1×10⁶ ATU of OPEN phage library were used toinfect >4×10⁸ B2H selection strain cells and the resulting transformantswere plated on a series of NM/CCK medium plates containing 50 μM IPTG,40 mM 3AT, and 60 μg/mL streptomycin, 50 μM IPTG, 60 mM 3AT, and 80μg/mL streptomycin, 0 μM IPTG, 40 mM 3AT, and 60 μg/mL streptomycin or 0μM IPTG, 60 mM 3AT, and 80 μg/mL streptomycin. For each target site, theinventors picked colonies from the highest stringency plates thatyielded colonies. For one site (EG382R), 4.25×10⁸ ATU of OPEN phagelibrary were used to infect >10⁹ B2H selection strain cells and theresulting transformants were plated on a 245×245 mm NM/CCK medium platecontaining parallel gradients of 3-AT and streptomycin ranging from 0 mMto 80 mM and 0 μg/mL to 100 μg/mL, respectively. Colonies were pickedfrom the highest stringency edge of the plate.

Construction of Modularly Assembled Zinc Finger Arrays.

Modularly assembled zinc finger arrays were assembled using the ZincFinger Consortium Modular Assembly Kit v1.0 as previously described(Wright et al., 2006). For each target half-site in the EGFP reportergene, the inventors assembled three-finger arrays using modules from theBarbas, Sangamo, and Toolgen archives but they did not mix and matchmodules across platforms because: (1) the Barbas group does not suggestuse of their modules with others (Mandell and Barbas, 2006), (2) theToolgen group discovered that their human zinc fingers worked best withone another but not as well with other engineered modules (Bae et al.,2003), and (3) the Sangamo modules were designed to befinger-position-specific and have non-canonical linkers joining themthat differ from the TGEKP linker used by the Barbas and Toolgen modules(Liu et al., 2002).

ZFN Expression Vectors.

All zinc finger arrays were expressed as ZFNs using the Zinc FingerConsortium mammalian expression vector pST1374 (Wright et al., 2006).Zinc finger arrays were excised directly from B2H expression vectors onan XbaI/BamHI fragment and cloned into pST1374. In this configuration,zinc finger arrays are joined to the FokI nuclease domain by a fouramino acid linker of sequence LRGS.

Human Cell-Based EGFP-Disruption Assay.

293.EGFP cells were transfected in triplicate in 24-well plates usingcalcium phosphate precipitation as previously described (Cathomen etal., 2001). Transfection cocktails included 300 ng each of a CMVpromoter-controlled zinc finger nuclease expression vector, 100 ngpDS.RedExpress (Clontech, Mountain view, CA) and pUC118 to 1.5 μg. 600ng of pRK5.SceI plasmid (Alwin et al., 2005), which expresses themeganuclease IScel, was used in place of GFP-ZFN-encoding plasmids fornegative controls. 50,000 cells were analyzed by flow cytometry two andfive days post-transfection to determine the percentage of EGFP-negativecells. The number of REx-positive cells at day 2 was used to normalizefor transfection efficiency.

CEL I Nuclease Assay for NHEJ-Mediated Mutation.

2×10⁶ human Flp-In T-REx 293 cells (Invitrogen) were transfected withpairs of ZFN-encoding plasmids (100 or 250 ng of each ZFN-encodingplasmid) using Lipofectamine 2000 (Invitrogen). Genomic DNA was isolatedfrom nuclease-treated cells at 3 days post-transfection using the QIAgenBlood Mini kit. Limited-cycle PCR (24 cycles) was performed usingPlatinum PCR SuperMix Hi-Fidelity (Invitrogen) or its equivalentconstituent components (Invitrogen) with 50 ng of genomic DNA astemplate, 8 μCi of each [α³²P]-dATP and dCTP, 1 μM each of gene-specificprimers and 1.25 μl DMSO in a 25 μl reaction volume. PCR products werecleaned up using Sephadex G-50 columns (Roche) and thenmelted/re-annealed using the following conditions: 95° C. for 10 min;95° C. to 85° C. cooling at a rate of −2° C./sec; 85° C. to 25° C.cooling at a rate of −0.1° C./sec; rapid cool to 4° C. Re-annealed PCRproducts were diluted 1:3.75 in a buffer of 20 mM Tris-HCl (pH 8.8), 2mM MgSO₄, 60 mM KCl, 0.1% Triton X-100 and treated with 1 μl CEL Ienzyme (Surveyor nuclease S; Transgenomic) and 1 μl Surveyor Enhancer S(Transgenomic) in a 15 μl reaction incubated at 42° C. for 20 min.Products were visualized by electrophoresis on a 0.8 mm thick, 10% 1×TBEpolyacrylamide gel which was dried down and exposed overnight to aphosphorimaging screen.

Gene Targeting Assays.

2×10⁶ Flp-In T-REx 293 cells were transfected with pairs of plasmidsexpressing ZFNs (7.5 μg of each ZFN-encoding plasmid) and 50 μg of donorplasmid using nucleofection with solution V and program Q001 (Amaxa).2×10⁶ K562 cells were transfected with ZFN expression plasmid pairs (5or 7.5 μg of each) and matched donor construct (25 or 50 μg donorplasmid) using nucleofection with solution V and program T-16. GenomicDNA was harvested 3 or 4 days post-transfection for 293 or K562 cells,respectively, using a QIAgen Blood Mini kit. Transfection efficiencieswere monitored by including a GFP-encoding plasmid in each transfectionand determining the percentage of GFP-positive cells by flow cytometryone day post-transfection. For experiments in which cells were arrestedin G2 phase, 0.2 μM vinblastine was added 24 hours post-transfection andthen removed by washing three times with phosphate buffered saline 14-18hours later. Limited-cycle PCR assays (24 cycles) were performed usingPlatinum PCR SuperMix Hi-Fidelity (Invitrogen) or its equivalentconstituent components (Invitrogen) with 4 ng genomic DNA, 8 μCi of each[α³²P]-dATP and dCTP, 1 μM each of gene-specific primers and 1.25 μlDMSO in a 25 μl reaction. Purified PCR product was digested with 25units SalI or 10 units BsrBI restriction enzyme for 2 hours and theresulting products were visualized by electrophoresis on a 10% 1×TBEpolyacrylamide gel. This gel was dried down and exposed overnight to aphosphorimaging screen. Quantification of bands was performed usingQuantity One software (Bio-Rad).

For Southern blots, 15 μg of genomic DNA (15 μg) was digested with MscIand SalI restriction enzymes for 20 hrs, electrophoresed in 0.8%tris-acetate agarose gels (100 mM Tris-HCl, 10 mM EDTA, pH 8.0 withacetic acid), and transferred to Zeta-probe nylon membrane (BioRad)using 25 mM sodium phosphate (pH 6.5) according to the procedure ofSouthern (Southern, 1975) as modified for use with the Turbo-Blotdownward transfer apparatus. The VEGF-A DNA probe was generated by PCRamplification of a cloned human VEGF-A DNA template which wassubsequently labeled (25 ng) with [α³²P]-dCTP) using Rediprime II randompriming reagents (Amersham). Following hybridization (20 hrs) at 65° C.in 5 mL ExpressHyb solution (Clontech), the filters were washed with0.1×SSC/0.1% SDS at 65° C. (2 hrs), blotted dry and exposed to aphosphorimager screen and/or film. The filters were scanned in theTyphoon 8600 phosphoimager and relative band intensities were quantifiedby volume analysis using ImageQuant software (GE Healthcare/Amersham).

Sequencing of Modified Genomic Alleles.

The region encompassing each potential ZFN cleavage site was amplifiedfrom genomic DNA isolated from populations of human Flp-In T-REx 293 orK562 cells that had been transfected with ZFN expression plasmids aloneor with ZFN expression and donor plasmids. PCR conditions for theseamplifications were the same as those used for the CEL I (for assessingNHEJ events) or limited-cycle PCR/restriction digest (for assessing genetargeting events) assays but with all components doubled to a finalvolume of 50 μl. CF877 was amplified using primers OK1711 and 1713. PCRreactions were purified using the QIAgen Minelute PCR Purification kitand eluted with 15 μl 0.1× EB buffer (QIAgen). PCR fragments were clonedinto the pCR4Blunt-TOPO plasmid using the Zero Blunt TOPO PCR CloningKit for Sequencing (Invitrogen). The TOPO cloning reaction used 4 μlpurified PCR product, 1 μl salt solution and 1 μl TOPO vector. 2 μl ofTOPO reaction were transformed into One Shot Mach1-T1 chemicallycompetent cells (Invitrogen) or chemically competent Top10 cells(Invitrogen) and plated on LB plates containing 50 μg/ml kanamycin.Plasmid DNAs from transformants were sequenced with a primer designed tobind internal to the PCR product.

Tobacco Transformation and Assay for Mutations.

The transformation of tobacco protoplasts by electroporation was carriedout as previously described (Wright et al., 2005). Plasmids introducedinto protoplasts (10 μg each) included those expressing ZFNs thatrecognize the left (pRW242) and right (pRW246) half sites of target 2163in SuRA. Note that these constructs do not express the heterodimericvariants of FokI endonuclease. Also transformed into protoplasts was aplasmid expressing neomycin phosphotransferase (NPTII) (pDW998). TheCaMV 35S promoter was used to drive expression of both the ZFNs andNPTII. Plasmid DNAs were linearized with BglII prior to transformation.Protoplasts were allowed to recover and then selected for kanamycinresistance and regenerated into plantlets as previously described(Wright et al., 2005).

DNA was prepared from tissue harvested from individual plantlets usingthe Epicentre MasterPure Plant Leaf DNA Purification Kit following themanufacturer's directions. An initial PCR screen for mutations at thesite of ZFN cleavage was performed using primers to amplify a 445 bpfragment from both the SuRA and SuRB loci. PCR was performed with 100 ngof genomic DNA and the following PCR conditions: 94° C. 2 min, followedby 34 cycles of 94° C. 15 sec, 61° C. 15 sec, 72° C. 30 sec, and then72° C. for 5 min. The reactions were run out on a 0.8% agarose gel,purified using a QIAgen QlAquick Gel Extraction kit, and sequenced. Theresulting sequences were examined for double peaks, which not onlyindicate sequence differences between SuRA and SuRB, but also identifypotential insertion/deletion events at the ZFN cleavage site in eitheror both loci.

DNA from candidate mutants was then PCR amplified using a set of nested,allele-specific primers in two consecutive PCR reactions to confirm themutation and determine if it occurred in SuRA or SuRB. The primaryreaction amplified a 2.15 kb fragment using approximately 100 ng ofgenomic DNA as template and primers for SuRA and SuRB. The second PCRreaction amplified a 2 kb fragment using 1 μl of the primary PCRreaction as template and primers SuRA and SuRB. All PCR reactions wereperformed using a Clontech Advantage cDNA Polymerase kit and thefollowing PCR conditions: 94° C. 1 min, followed by 34 cycles of 94° C.30 sec, 66° C. 30 sec, 68° C. 3 min, and then 68° C. for 5 min. Thereactions were run out on a 0.8% agarose gel, purified with a QIAgenQlAquick Gel Extraction kit and sequenced with primer DVO4462.

ZFN Toxicity Assays.

ZFN expression plasmids and donor templates were transfected into K562cells using nucleofection as described above. For these experiments, 5μg of each ZFN expression plasmid, 25 μg of donor, and 15 ng of pmaxGFP(encoding a GFP variant; Amaxa) were included in each transfection. Forcontrols, the inventors transfected: (1) 10 μg of plasmid encodingI-SceI meganuclease (Porteus and Baltimore, 2003) with 25 μg pUC118 and15 ng of pmaxGFP, (2) 10 μg of plasmid encoding CAD (caspase-activatedDNase) protein (Pruett-Miller et al., 2008) with 25 μg pUC118 and 15 ngof pmaxGFP, or (3) 35 μg of pUC118 and 15 ng of pmaxGFP. Cells wereassayed for GFP expression at post-transfection days 1 and 7 with aFACScan cytometer. GFP ratios shown in FIG. 4J (green bars) werecalculated using the formula:

$\frac{\begin{pmatrix}{{\% \mspace{14mu} {GFP}} + {{in}\mspace{14mu} {ZFN}\text{-}{transfected}\mspace{14mu} {cells}\mspace{14mu} {on}\mspace{14mu} {day}\mspace{14mu} 7\text{/}}} \\{{\% \mspace{14mu} {GFP}} + {{in}\mspace{14mu} {ZFN}\text{-}{transfected}\mspace{14mu} {cells}\mspace{14mu} {on}\mspace{14mu} {day}}}\end{pmatrix}}{\begin{pmatrix}{{\% \mspace{14mu} {GFP}} + {{in}\mspace{14mu} {pUC}\text{-}{transfected}\mspace{14mu} {cells}\mspace{14mu} {on}\mspace{14mu} {day}\mspace{14mu} 7\text{/}}} \\{{\% \mspace{14mu} {GFP}} + {{in}\mspace{14mu} {pUC}\text{-}{transfected}\mspace{14mu} {cells}\mspace{14mu} {on}\mspace{14mu} {day}}}\end{pmatrix}}$

In addition, genomic DNA was harvested using a QIAgen Blood Mini Prepkit on post-transfection days 4 and 7 and assayed for gene targetingusing the limited-cycle PCR/restriction digest assay as described above.Gene targeting ratios shown in FIG. 4J (purple bars) were calculated bydividing the gene targeting rate on day 7 by the gene targeting rate onday 4. All assays (both GFP and gene targeting) were performed on atleast three-independent samples and t-tests of significance wereperformed by comparing experimentally determined ratios to a fixed ratiovalue of 1 (i.e., no change in value).

Example 2 Results

OPEN: A Rapid and Robust Strategy for Engineering Zinc-Finger Arrays.

Previous work has suggested that context-dependent DNA-binding effectscan occur within multi-finger arrays (i.e., the binding activity of onefinger may be influenced by neighboring fingers) (Elrod-Erickson et al.,1996; Isalan et al., 1998; Wolfe et al., 1999). Failure to considerthese context-dependent effects is a likely reason for the very lowsuccess rates observed with modular assembly strategies (Ramirez et al.,2008) which treat individual finger domains as independent units.Various groups have proposed strategies for engineering zinc-fingerarrays that identify combinations of fingers from randomized librariesthat work well together, thereby accounting for context-dependence.

Zinc-finger arrays engineered using these approaches possess highDNA-binding affinities and specificities (Greisman and Pabo, 1997; Hurtet al., 2003; Isalan et al., 2001) and function with high activities andlow toxicities when expressed as ZFNs in human cells (Cornu et al.,2008; Pruett-Miller et al., 2008). However, all of these strategiesrequire the construction of multiple randomized libraries typically >10⁸in size and are therefore impractical for routine use by all but a fewlaboratories that possess the required expertise.

The new OPEN method for engineering zinc-finger arrays considerscontext-dependent effects but also eliminates the need for specializedexpertise in the construction and interrogation of multiple, very largerandomized libraries. The practice of OPEN requires an archive ofpre-selected zinc-finger pools, each targeted to a different three basepair subsite at one of three finger positions within a three-fingerarray (FIGS. 1A and 1B). Each finger pool contains a mixture ofzinc-fingers (maximum of 95) that is obtained by performing lowstringency selections from very large zinc-finger libraries >2×10⁸ insize (see above). To use OPEN to identify a multi-finger array thatbinds to a given target DNA site, appropriate finger pools from thearchive are recombined to create a small single library of variantswhich is then interrogated using plates containing gradients ofselective agents (FIG. 1B). Because each finger pool is composed of nomore than 95 different members, the combinatorial diversity of thelibrary is relatively modest (95³=8.6×10⁵ for a three-finger domain).Selections are performed using a simple bacterial two-hybrid (B2H)system in which binding of a zinc-finger domain to its cognate sitetriggers the expression of selectable marker genes (FIG. 1C) (Hurt etal., 2003; Joung et al., 2000). The inventors reasoned that this overallapproach would identify combinations of fingers that work well together(thereby accounting for context-dependent effects on DNA-binding) whileavoiding the need for the end-user to interrogate very large randomizedlibraries—a technically demanding and labor-intensive step.

Construction of an Initial Archive of OPEN Finger Pools.

Fully enabling the OPEN method requires an archive of 192 finger pools(64 possible three base pair target subsites for each position in athree finger protein). As an initial step toward full implementation ofOPEN, pools were created that recognize subsites of the form 5′-GXX or5′-TXX (where X is any base). GXX subsites were chosen to allow directcomparison of OPEN-generated three-finger domains with those made bymodular assembly (Bae et al., 2003; Liu et al., 2002; Mandell andBarbas, 2006). TXX subsites were of interest because very few fingermodules that bind to these sites have been reported (Mandell and Barbas,2006). Using the approach outlined in FIG. 1A, finger pools were createdfor a total of 66 subsites (all possible 48 GXX subsites and 18 TXXsubsites). For each finger pool, the inventors inoculated 95 survivingcolonies into 96-well plates for growth and isolation ofzinc-finger-encoding plasmid DNA (FIG. 1A). Even with this partial setof finger pools, OPEN can be used to target ˜4% of all 262,144 potentialnine base pair target sites, thereby enabling researchers on average tofind approximately five full ZFN sites in any given kb of sequence. Tosimplify the identification of potential ZFN sites that can be targetedwith the initial archive of OPEN finger pools, the inventors created amodified version of their pre-existing ZiFiT software which wasoriginally designed for use with the modular assembly zinc-fingerengineering method (Sander et al., 2007; Wright et al., 2006). This newversion, Zinc Finger Targeter, V 2.0 (available on the world-wide-web atzincfingers.org), scans an input sequence and identifies allthree-finger ZFN sites that can be targeted by OPEN.

Comparing ZFNs Made by Modular Assembly and OPEN Methods.

To compare the efficacy of the OPEN method with the well-describedmodular assembly approach, the inventors constructed multi-fingerdomains against five potential ZFN target sites in EGFP (ten“half-sites”; see FIG. 2A) using both strategies. Modularly assembledarrays were made using finger modules from three different archives (seeabove). OPEN selections were used to engineer zinc finger arrays for theten EGFP half-sites as described in Experimental Procedures. All 10 OPENselections yielded zinc-finger arrays which closely resemble oneanother), a result which suggests that the selections identified thebest combinations of fingers for each target site.

DNA-binding activities of the modularly assembled and OPEN-selectedzinc-finger arrays targeted to EGFP sequences were assessed using aquantitative version of the B2H assay. Previous studies have shown thatzinc-finger arrays with high affinities and specificities activatetranscription more than three-fold in the B2H system (Hurt et al.,2003). As shown in FIG. 2B, all of the modularly assembled zinc-fingerarrays tested on the 10 target half-sites failed to activatetranscription by more than threefold. Western blotting verified that allmodularly assembled zinc-finger arrays were expressed in these assays(data not shown). By contrast, OPEN selections yielded at least one—inmost cases, many—zinc-finger array that activated transcription in theB2H by more than three-fold for nine of the 10 target half-sites (FIG.2B). Although half-site EG502L did not yield OPEN zinc-finger arrayswhich activated by more than three-fold, the inventors note that thebasal level of transcription from the B2H reporter bearing this site washigh, a situation which can artifactually lower the apparentfold-activation observed.

The EGFP zinc-finger arrays made by modular assembly and OPEN were nexttested as ZFNs in human cells. Previous studies have shown that ZFNs caninduce (via error-prone NHEJ) targeted insertions and deletions intohuman genes with high efficiency (Lombardo et al., 2007; Miller et al.,2007). Therefore, the inventors tested the activities of ZFNs using ahuman cell-based EGFP-disruption assay in which ZFN-induced DSBs lead tocoding sequence alterations of a chromosomally integrated EGFP reportergene (FIG. 2C). For each of the five full EGFP ZFN target sites, two orthree pairs of modularly assembled ZFNs and four pairs of OPEN-selectedZFNs were chosen for testing. As shown in FIG. 2D, modular assemblyyielded ZFN pairs with activities above background for only one of thefive sites (EG502). By contrast, OPEN selection yielded active ZFN pairsfor four of the five full ZFN target sites (EG223, EG292, EG382, andEG502). Although both methods yielded active ZFN pairs for the EG502site, the pairs made by OPEN were significantly more active than thepairs made by modular assembly (FIG. 2D). Control Western blotexperiments verified the expression of all ZFNs tested (data not shown).The inventors speculate that the EG568 ZFNs may be inactive due to theeffects of methylation or chromatin at that site in the chromosomallyintegrated EGFP reporter because one of the modularly assembled and allof the OPEN ZFN pairs showed activity in an episomal-based version ofthis same assay (data not shown).

OPEN Selection of Zinc-Finger Arrays that Bind to Sequences inEndogenous Human and Plant Genes.

To further test the efficiency of the OPEN approach for engineeringmulti-finger arrays, ZiFiT 2.0 was used to identify 14 potential ZFNtarget sites in three endogenous human genes (CFTR, HoxB13, VEGF-A) andone endogenous plant gene (SuRA from Nicotiana tobacum) (FIG. 3A). TheVEGF-A gene plays a critical role in angiogenesis, and the inventorstargeted six different sites located within regions of open chromatin inits promoter (VF2468, VF2471, VF3537, VF3540, VF3542, and VF3552). TheHoxB13 gene has been implicated as an important biomarker for resistanceof breast cancers to the anti-hormonal drug tamoxifen (Ma et al., 2006;Ma et al., 2004), and the inventors targeted five sites (HX500, HX508,HX587, HX735, and HX761) within its first coding exon and one site(HX2119) within its last coding exon. The CFTR gene encodes a chloridechannel that is mutated in people with cystic fibrosis (Welsh et al.,2001) and the inventors targeted a single site in exon 10 (CF877) thatis positioned within 100 base pairs of the ΔF508 deletion. The tobaccoSuRA and SuRB genes are highly similar and both encode acetolactatesynthase, an enzyme which carries out the first step in branched chainamino acid synthesis and which is inhibited by several herbicides (Leeet al., 1988; McCourt et al., 2006); the inventors targeted a singlesite (SR2163) that is present in both the SuRA and SuRB genes.

OPEN selections were performed for the 28 different nine base pairsites, which together constitute the “half-sites” of the 14 full ZFNtarget sites. Twenty-five of the 28 OPEN selections yielded zinc-fingerarrays whose sequences closely resembled one another, again suggestingthat these selections identified the best combinations of fingers.However, one of these 25 selections (for half-site VF3540R) yieldedfingers with sequences that appeared to bind to an alternative site. For22 of the other 24 selections, the inventors obtained at least onezinc-finger array which activated lacZ expression by more thanthree-fold in the quantitative B2H assay; the two remaining selectionswere also deemed to be successful because their reporters possessed ahigh basal level of transcription which can artifually mask a highertrue fold-activation. Using the finger arrays, the inventors constructedZFN pairs for five full sites in VEGF-A, four full sites in HoxB13, onefull site in CFTR, and one full site in the tobacco SuRA/SuRB genes.

OPEN ZFNs Induce Highly Efficient Mutation of Endogenous Human and PlantGenes.

To test whether OPEN ZFNs can induce NHEJ-mediated mutations at thehuman VEGFA, HoxB13, and CFTR genes, the inventors employed a previouslydescribed mutation detection assay which uses the CEL I nuclease todetect ZFN-induced insertions or deletions (Lombardo et al., 2007;Miller et al., 2007). In this assay, limited-cycle PCR is used toamplify a locus of interest from the genomic DNA of a population ofhuman cells transfected with ZFN expression vectors (FIG. 3B). Theresulting PCR product is denatured and re-annealed and heteroduplex DNAwill form if mutated alleles are present in the population. These DNAfragments can be cleaved at the site of mismatch by the CEL I enzymeinto smaller products of predictable size. Using this assay, theinventors found that all four VF2468 and all four VF2471 ZFN pairsinduced detectable mutation of the endogenous VEGF-A gene promoter inhuman 293 cells (FIG. 3C). None of the VF3537, VF3542, or VF3552 ZFNpairs induced detectable levels of mutation. In addition, mutationscould be detected at the endogenous HoxB13 gene in 293 cells for all ZFNpairs tested at the HX587, HX735 and HX761 sites (FIG. 3D). No evidenceof mutations was observed for any pairs tested at the HX508 site. DNAsequencing of HoxB13 alleles from cells modified by the HX587 and HX761confirmed the presence of mutations at the appropriate ZFN cleavage site(data not shown and below). Due to the presence of a polymorphism in themiddle of the CF877 site in both human 293 and K562 cells, the inventorscould not assess the activity of the CF877 ZFN pairs (data not shown).

Although previous reports suggested that the CEL I assay can be used toquantify mutations in a population of alleles (Lombardo et al., 2007;Miller et al., 2007), in the inventors' hands this assay did not behavein a reproducibly quantitative manner. Therefore, they instead assessedthe frequency of mutations induced by the OPEN ZFNs using a combinationof limited-cycle PCR and DNA sequencing (see above). HX587 ZFN pair Band the CF877 ZFN pair were chosen for quantification. Insertion anddeletion mutations were observed at the expected location in the middleof the HX587 site with an average frequency of 9.6% (FIG. 3E). For theCFTR locus, insertions at the CF877 cleavage site were observed with anaverage frequency of 1.2% (FIG. 3F), providing evidence that this pairof ZFNs was active in human cells. These mutation frequencies induced byOPEN ZFNs are comparable to rates of NHEJ-mediated mutation induced byZFNs targeted to the human IL2Rγ gene in a previous study (Lombardo etal., 2007).

To test whether OPEN ZFNs function effectively in cells other thanhuman, the inventors sought to modify an endogenous gene in plants.Tobacco protoplasts were transformed with a construct encoding theSR2163 ZFN pair as well as a construct that expresses kanamycinresistance (to identify cells successfully transformed with exogenousDNA). Kanamycin-resistant cells were selected and regenerated intoindividual plants, and the SR2163 site in each plant was examined forevidence of cleavage in both SuRA and SuRB using PCR amplification andDNA sequencing. Among 66 transgenic plants surveyed, three had mutationsin SuRA, all of which were deletions of a single base (FIG. 3G). In oneplant, both alleles of SuRA had the same deletion. This frequency ofmutagenesis by NHEJ (˜2% of potential target alleles) is comparable towhat the inventors observed for other OPEN ZFNs in human cells.

OPEN ZFNs Induce Highly Efficient Gene Targeting of Endogenous HumanGenes.

The inventors next tested whether their OPEN ZFNs could be used toinduce high efficiency gene targeting at the endogenous VEGF-A gene inhuman 293 cells. For all gene targeting experiments performed, theinventors used a single ZFN pair for each of the two different targetsites: VF2468 pair C and VF2471 pair B. They also constructedappropriate “donor templates” for the VF2468 and VF2471 sites which eachcontain 1.5 kb of genomic DNA sequence centered on the cleavage site andintroduce an 11 bp insertion encoding a SalI restriction site at thecenter of the cleavage site (FIG. 4A). 293 cells were transfected withexpression plasmids encoding ZFN expression vectors and the appropriatedonor plasmid and the frequency of successful gene targeting wasmeasured three days post-transfection using a previously describedlimited-cycle PCR/restriction digest assay (see above) (Urnov et al.,2005). Mean gene targeting frequencies of 2.95% and 2.88% were obtainedwith the VF2468 and VF2471 ZFN pairs, respectively, with controlsdemonstrating the requirement for both the donor plasmid and ZFNexpression plasmids (FIG. 4B).

The inventors also tested whether their VF2468 and VF2471 ZFN pairscould mediate high efficiency gene targeting in K562 cells, anotherhuman cell line. To perform a side-by-side comparison with a previouslypublished IL2Rγ-specific ZFN pair (Miller et al., 2007), the inventorscloned DNA sequences encoding these nucleases into the ZFN expressionvectors and also constructed a donor plasmid containing 1.5 kb ofgenomic IL2Rγ sequence centered on a translationally silent pointmutation that creates a BsrBI restriction site at the ZFN cleavage site(Urnov et al., 2005) (FIG. 4A). Mean gene targeting efficiencies of7.7%, 4.5% and 4.1% were observed with the VF2468, VF2471, and IL2RγZFNs, respectively (FIG. 4C). The IL2Rγ ZFNs induced gene targetingrates similar to those previously published (Urnov et al., 2005).Increasing the concentrations of ZFN expression vectors and donorplasmid did not appreciably improve gene targeting efficiencies for anyof the three pairs tested (data not shown). Control experimentsdemonstrated that efficient gene targeting required the presence of boththe donor construct and ZFN expression vectors (FIG. 4C). To confirmthat the results accurately quantified gene targeting frequencies, theinventors performed both PCR-based assays and Southern blotting ongenomic DNA harvested from cells modified by VF2468 and VF2471 ZFNs. Thetwo methods gave similar results, but the PCR-based assay tended tounderestimate gene targeting rates relative to Southern blots (FIGS. 4Dand 4E). They conclude that the IL2Rγ-specific ZFNs and the OPEN VEGF-AZFNs induce comparable gene targeting frequencies in human cells.

In a previous study, higher rates of gene targeting were observed ifcells were transiently arrested in G2 phase with vinblastine (Urnov etal., 2005). The inventors also observed ˜8-fold higher rates of genetargeting in vinblastine-treated K562 cells assayed at four daysposttransfection with the limited-cycle PCR/restriction digest assay:54%, 37%, and 44% mean gene targeting efficiencies with VF2468, VF2471,and IL2Rγ ZFNs, respectively (FIG. 4F). Notably, however, vinblastinetreatment greatly reduced the number of viable cells. Sequencing ofVEGF-A or IL2Rγ alleles amplified from genomic DNA of thesevinblastine-treated cells revealed gene targeting events at the expectedlocations and with frequencies that matched well with the results oflimited-cycle PCR assays (FIGS. 4G-4I). For all three target sites, theinventors also observed high frequencies of insertion and deletionevents at the ZFN cleavage sites, presumably caused by error-prone NHEJ.

Unexpectedly, the inventors found that 1.8% and 9.0% of the alleles fromcells treated with VF2468 and IL2Rγ ZFNs, respectively, containedevidence of both gene targeting and NHEJ-mediated insertion events at asingle allele. They note that these doubly altered alleles could only befound by sequencing and would not have been detected in previouslypublished studies that used PCR-based or Southern blot assays to detectgene targeting events (Lombardo et al., 2007; Urnov et al., 2005).

Toxicity Profiles of OPEN ZFNs.

Some three-finger ZFNs made by modular assembly or rational design cancause cellular toxicity, an effect most likely due to unintended,off-target cleavage events (Cornu et al., 2008; Porteus and Baltimore,2003). By contrast, optimized IL2Rγ-targeted ZFNs, which possessfour-finger arrays, cause minimal toxicity (Miller et al., 2007; Urnovet al., 2005). The inventors therefore wished to compare the relativetoxicities of the three-finger OPEN VEGF-A ZFNs with the four-fingerIL2Rγ ZFNs. Cell survival assays were conducted in which K562 cells weretransfected with ZFN expression vector pairs, an appropriately matcheddonor plasmid, and a plasmid expressing GFP. GFP-positive cells wereassayed 1 and 7 days post-transfection and the percentage of genetargeting events was assessed 4 and 7 days post-transfection. Previousstudies have demonstrated that toxic ZFNs reduce the percentage ofGFP-positive cells (Cornu et al., 2008; Pruett-Miller et al., 2008) andthe percentage of cells that have undergone a gene targeting event(Porteus and Baltimore, 2003) over time. All three ZFN pairs showedsignificant reductions in the relative number of GFP-positive cells bypost-transfection day 7, with the IL2Rγ and VF2471 ZFN pairs exhibitingsimilar levels of toxicity and the VF2468 ZFN pair exhibiting a greaterlevel of toxicity which rivaled that induced by the control CAD(caspase-activated DNase) protein (FIG. 4J, green bars). As expected,analogous decreases were also observed in the percentage of genetargeting events measurable in the cell populations (FIG. 4J, purplebars).

The use of obligate heterodimeric FokI nuclease domain variants cansignificantly reduce ZFN-associated toxicity (Miller et al., 2007;Szczepek et al., 2007). The inventors examined the toxicities of theSangamo IL2Rγ and the OPEN VF2468 and VF2471 ZFNs harboring thesevariant FokI domains. Both the variant IL2Rγ ZFN pair and the variantVF2471 ZFN pair showed no significant toxicity as judged by the GFPtoxicity assay (FIG. 4J, green bars). In addition, the variant VF2468ZFN pair revealed minimal toxicity (FIG. 4J) of a level comparable tothat of I-SceI, a highly specific meganuclease used as a “nontoxic”control in previous studies (Porteus and Baltimore, 2003). Comparableeffects were also observed for these variant nucleases when examiningthe relative percentage of gene targeting events in the cell populations(FIG. 4J, purple bars). The absolute gene targeting frequencies inducedby the three variant ZFN pairs were comparable at day 4 (FIG. 4K),demonstrating that the absence of observable toxicity is not due to lackof ZFN activity. The inventors conclude that the three-finger OPEN ZFNscan exhibit toxicity profiles comparable to that of an optimizedfour-finger ZFN, particularly when they are expressed in an obligateheterodimer framework.

OPEN ZFNs Mediate Stable Multi-Allelic Changes in Human Cells.

To test whether OPEN ZFN-induced gene targeting events are stablymaintained, limiting dilution cloning was used to isolate single cellclones from K562 cell populations that had been modified withhomodimeric VF2468 or VF2471 ZFNs. 30 days following transfection, twovinblastine-treated K562 cell populations showed high frequencies ofgene targeting: 45% and 26% with the VF2468 and VF2471 ZFN pairs,respectively (FIG. 5A). Of note, the VF2468 ZFN-treated cells alsoshowed evidence of a 625 bp deletion (FIG. 5A, blue asterisks), afinding confirmed by sequencing of alleles from these cells (data notshown). FISH analysis indicated that K562 cells harbor four alleles ofthe VEGF-A gene (FIG. 5B), and the inventors therefore wished todetermine how many of these alleles were altered in individual cellswithin the population. Using dilution cloning, the inventors isolated 27and 28 single cell clones for the VF2468 and VF2471 ZFN-treated cellpopulations, respectively. Genotype analysis of individual clones fromthe VF2471 ZFN-treated cells revealed clones in which no, one, two, orthree VEGF alleles had undergone a gene targeting event (FIG. 5A, lowerright panel). Individual clones from VF2468 ZFN-treated cells harboredno, one, three, or four alleles that had undergone a gene targetingevent with some of the clones also containing the 625 bp deletion (FIG.5A, lower left panel). All individual cell clones were genotyped morethan forty days post-transfection, demonstrating that gene targetingevents the inventors observed are stably maintained. As a control, theyperformed FISH analysis on three clones in which all VEGF-A alleles ineach cell had undergone the VF2468 ZFN-induced gene targeting event andconfirmed the continued presence of four copies of VEGF-A per cell (FIG.5B). Taken together, these results demonstrate that OPEN ZFNs can beused to induce permanent alterations in as many as four alleles in asingle human cell.

Using Zinc Finger Nucleases (ZFNs) constructed by the methods above, theinventors generated ZFN pairs that bind to the CFTR gene and createdouble-strand breaks (DSBs) at a site 123 nucleotides from the ΔF508deletion (Tables 1-2). To assess whether these ZFNs could introducetargeted DSBs within the endogenous human CFTR gene, the inventorslooked for the introduction of small insertions or deletions (indels)which result from error-prone repair by non-homologous end joining(NHEJ). When they introduced plasmids encoding ZFNs by nucleofection,the rate of NHEJ-mediated indels observed was 1.2% as assessed by DNAsequencing.

TABLE 1A  CFTR ZINC FINGER DOMAINS (LEFT ARM) GTG GAA TTA OZ107 CF877L2- RKHIL QGGNLVR QQTGLAA 1.92 0.58 step DT (SEQ ID (SEQ ID gra- (SEQNO: 4) NO: 5) dient ID NO: 3) OZ108 CF877L 2- RKSVL QGGNLVR QTTGLKS 2.310.51 step LV (SEQ ID (SEQ ID gra- (SEQ NO: 7) NO: 8) dient ID NO: 6)OZ109 CF877L 2- RTSSL RREHLTR QPTGLTA 2.83 0.20 step KR (SEQ ID (SEQ IDgra- (SEQ NO: 10) NO: 11) dient ID NO: 9) OZ110 CF877L 2- RNFIL QGGNLVRQVNGLKA 1.61 0.15 + step QR (SEQ ID (SEQ ID gra- (SEQ NO: 13) NO: 14)dient ID NO: 12) OZ111 CF877L 2- RKGVL QGGNLVR QQTGLNV 2.20 1.90 + stepRI (SEQ ID (SEQ ID gra- (SEQ NO: 16) NO: 17) dient ID NO: 15) OZ112CF877L 2- RTSSL RREHLTR QPTGLTA 2.52 0.55 step KR (SEQ ID (SEQ ID gra-(SEQ NO: 19) NO: 20) dient ID NO: 18) OZ113 CF877L 2- RKSVL QGGNLVRQTTGLKS 1.61 0.20 step HN (SEQ ID (SEQ ID gra- (SEQ NO: 22) NO: 23)dient ID NO: 21) OZ114 CF877L 2- RNFIL QGGNLVR QQTGLAA 2.95 0.18 step QR(SEQ ID (SEQ ID gra- (SEQ NO: 25) NO: 26) dient ID NO: 24) OZ115 CF877L2- RNFIL QGGNLVR QVNGLKA 2.71 0.12 step QR (SEQ ID (SEQ ID gra- (SEQNO: 28) NO: 29) dient ID NO: 27) OZ116 CF877L 2- RRHVL QGGNLVR QQTGLNV2.53 0.38 step ER (SEQ ID (SEQ ID gra- (SEQ NO: 31) NO: 32) dient ID NO:30) OZ117 CF877L 2- RKSVL QGGNLVR QQTGLAA 3.87 0.33 step LV (SEQ ID(SEQ ID gra- (SEQ NO: 34) NO: 35) dient ID NO: 33) OZ118 CF877L 2- RNFILQGGNLVR QQTGLNV 3.55 0.08 step QR (SEQ ID (SEQ ID gra- (SEQ NO: 37)NO: 38) dient ID NO: 36)

TABLE 1B CFTR ZINC FINGER DOMAINS (RIGHT ARM) GAG TGG TTA OZ119 CF877R2- RQSNLSR RKEHLDI QMTGLNA 2.78 0.35 step (SEQ ID (SEQ ID (SEQ ID gra-NO: 39) NO: 40) NO: 41) dient OZ120 CF877R 2- RQSNLAR RKEHLVG QASGLNS2.53 0.01 step (SEQ ID (SEQ ID (SEQ ID gra- NO: 42) NO: 43) NO: 44)dient OZ121 CF877R 2- RQSNLSR RKEHLSI QRTGLTA 2.91 0.19 step (SEQ ID(SEQ ID (SEQ ID gra- NO: 45) NO: 46) NO: 47) dient OZ122 CF877R 2-TTHNLMR RADHLKV QGTGLRA 4.72 0.59 step (SEQ ID (SEQ ID (SEQ ID gra-NO: 48) NO: 49) NO: 50) dient OZ123 CF877R 2- TKHNLVR RREHLNI QTSGLTA5.03 0.63 step (SEQ ID (SEQ ID (SEQ ID gra- NO: 51) NO: 52) NO: 53)dient OZ124 CF877R 2- TKHNLVR RREHLNI QTSGLTA 4.41 0.20 step (SEQ ID(SEQ ID (SEQ ID gra- NO: 54) NO: 55) NO: 56) dient OZ125 CF877R 2-TKHNLVR RQEHLNI QPTGLKV 4.00 0.85 step (SEQ ID (SEQ ID (SEQ ID gra-NO: 57) NO: 58) NO: 59) dient OZ126 CF877R 2- TAHNLMR RREHLTI QMTGLNA2.57 0.28 step (SEQ ID (SEQ ID (SEQ ID gra- NO: 60) NO: 61) NO: 62)dient OZ127 CF877R 2- RMSNLDR RREHLTI QGTGLRA 1.60 0.02 step (SEQ ID(SEQ ID (SEQ ID gra- NO: 63) NO: 64) NO: 65) dient OZ128 CF877R 2-TTHNLMR RKEHLSI QMTGLNA 2.12 0.07 step (SEQ ID (SEQ ID (SEQ ID gra-NO: 66) NO: 67) NO: 68) dient OZ129 CF877R 2- RQSNLSR RKEHLDI QMTGLNA2.55 0.07 step (SEQ ID (SEQ ID (SEQ ID gra- NO: 69) NO: 70) NO: 71)dient OZ130 CF877R 2- RPHNLLR RADHLKV QTTGLNA 2.47 0.22 step (SEQ ID(SEQ ID (SEQ ID gra- NO: 72) NO: 73) NO: 74) dient OZ131 CF877R 2-KHSNLTR RREHLTI QPTGLRA 2.08 0.12 step (SEQ ID (SEQ ID (SEQ ID gra-NO: 75) NO: 76) NO: 77) dient OZ132 CF877R 2- RQSNLSR RSEHLAI QRVGLHA1.28 0.05 step (SEQ ID (SEQ ID (SEQ ID gra- NO: 78) NO: 79) NO: 80)dient OZ133 CF877R 2- KHSNLTR RADHLKV QNTGLHA 3.24 0.05 step (SEQ ID(SEQ ID (SEQ ID gra- NO: 81) NO: 82) NO: 83) dient OZ134 CF877R 2-RQSNLSR RNEHLVL QKTGLRV 3.71 0.19 step (SEQ ID (SEQ ID (SEQ ID gra-NO: 84) NO: 85) NO: 86) dient OZ135 CF877R 2- KHSNLTR RREHLTI QMTGLNA2.25 0.07 step (SEQ ID (SEQ ID (SEQ ID gra- NO: 87) NO: 88) NO: 89)dient OZ136 CF877R 2- KHSNLTR RREHLTI QMTGLNA 2.16 0.06 step (SEQ ID(SEQ ID (SEQ ID gra- NO: 90) NO: 91) NO: 92) dient OZ137 CF877R 2-RHSNLTR RQEHLNI QMTGLNA 2.26 0.04 step (SEQ ID (SEQ ID (SEQ ID gra-NO: 93) NO: 94) NO: 95) dient OZ138 CF877R 2- KKTNLTR RREHLTI QQTGLNV2.52 0.41 step (SEQ ID (SEQ ID (SEQ ID gra- NO: 96) NO: 97) NO: 98)dient OZ139 CF877R 2- KHSNLTR RKEHLSI QMTGLNA 2.74 0.03 step (SEQ ID(SEQ ID (SEQ ID gra- NO: 99) NO: 100) NO: 101) dient OZ140 CF877R 2-KHSNLTR RKEHLTI QRTGLSI 2.99 0.48 step (SEQ ID (SEQ ID (SEQ ID gra-NO: 102) NO: 103) NO: 104) dient OZ141 CF877R 2- KHGNLTR RREHLTI QQTGLNV2.87 0.08 step (SEQ ID (SEQ ID (SEQ ID gra- NO: 105) NO: 106) NO: 107)dient OZ142 CF877R 2- KHSNLTR RKEHLDI QMTGLNA 2.73 0.16 step (SEQ ID(SEQ ID (SEQ ID gra- NO: 108) NO: 109) NO: 110) dient

The inventors hypothesized that a viral vector could more efficientlydeliver ZFN pairs to airway epithelial cells and thereby improve theobserved indel rate. One ZFN was delivered by an adenovirus that alsoencoded an eGFP reporter gene; and the partner ZFN-encoding adenovirusencoded an mCherry reporter gene (FIG. 8). In both viral vectors, theZFNs were driven by CMV promoter with the fluorophores driven by the RSVpromoter. This approach enabled the inventors to use FACS to quantifythe number of cells that express both ZFNs in the pair. Usingmultiplicities of infection (MOI) of 250 for each virus, they deliveredZFNs to CF bronchial epithelial cells homozygous for the ΔF508 mutation.The inventors found that on average 40% of the cells weredoubly-positive for expression of the fluorescent reporter genes.Surveyor nuclease (CEL-1) assays of infected cells (without sorting)showed that ZFNs induced NHEJ-mediated indels with an average efficiencyof 6% of alleles.

To further improve the efficiency of ZFN-induced indel formation, theinventors have designed an adenoviral vector expressing a ZFN pairjoined by an intervening picornavirus T2A sequence. In addition, toextend results beyond introduction of targeted NHEJ-mediated indels, theinventors have constructed an adenoviral vector carrying a DNA donorrepair template for performing homologous recombination-based genetargeting and a Cerulean fluorescent reporter gene driven by RSV.Experiments are underway to transduce CF epithelia with ZFN and donorvectors to stimulate homologous recombination and to thereby correct theΔF508 locus. In summary, adenoviral delivery of ZFNs improves theefficiency of ZFN delivery to airway epithelial cells, and increases theNHEJ frequency near the ΔF508 locus of CFTR.

The inventors also screened the ZFN pairs for activity by expressingthem using adenoviral vectors in CFBE cells (an immortalized humanairway epithelial cell line homozygous for the ΔF508 mutation, CFBE4lo(CFBE) cells) using the surveyor (Cel I) nuclease assay as describedabove. As shown in Lane A, the combination of L1, R1 gave the greatestevidence of cutting activity (FIG. 7). The inventors have also generateda donor vector carrying a 1320 kb portion of wild-type CFTR sequence,and an engineered unique restriction site BspH1 into the donor, as wellas unique silent mutations (FIG. 9).

FIG. 11A depicts an adenoviral vector expressing the ZFN pair targetingthe CF877 site as a single expression cassette with an interveningpicornavirus T2A sequence. FIG. 11B shows the frequency of NHEJincreased in a dose-dependent manner from ˜10% at MOI of 50 to ˜29% atMOI of 250 in CFBE4lo-cells.

FIG. 12 shows a radioactive assay for homologous recombination (HR).K-562 cells were nucleofected (Amaxa T-16, soln V) with ZFNs and donorDNA or ZFNs only. The donor template is ˜1.5 kb and has a 5 bp insertionin spacer region between the ZFN binding site where the ZFNs cleave thegenomic DNA. This insertion (TGTCA) creates a unique BspH1 site in thedonor. The genomic DNA is harvested 4 days post nucleofection. PCR usingprimers binding outside of the donor template and radiolabeled dNTPs isfollowed by BspH I digestion. In the experiments with ‘+V’, 24 hrspost-nucleofection, the cells were treated with vinblastine (0.2 μM) for16 hrs. The presence of fragments at 1136 and 504 bp indicates HR. HRwas confirmed by DNA sequencing.

FIG. 13 shows a radioactive assay for homologous recombination (HR) inepithelia. CFBE (CF ΔF508/ΔF508 bronchial epithelial cells) wereco-transduced with adenoviral vectors expressing the ZFN pair (AdZ) andthe homologous recombination donor (AdD) or with AdZ only. The donortemplate is ˜1.5 kb and has a 5 bp insertion in spacer region betweenthe ZFN binding sites. This insertion (TGTCA) creates a unique BspH1site in the donor. The genomic DNA is harvested 4 days posttransduction. PCR using primers binding outside of the donor templateand radiolabeled dNTPs is followed by BspH I digestion. The presence offragments at 1136 and 504 bp indicates HR. Direct DNA sequencing alsodocumented HR in cells receiving AdZ and AdD, but not AdZ alone.

Together, FIGS. 11-13 show that delivery of the ZFN pair targeting exon10 of CFTR by adenoviral vector dramatically increases the frequency ofdouble stranded breaks as assessed by identification of insertions anddeletions at the site of cutting by direct sequencing. In addition,these data now provide evidence of homologous recombination at the CFTRexon 10 site, both in in K562 cells transduced with plasmid vectors andelectroporation, and in CF epithelial cells transduced with adenoviralvectors to deliver the donor and the ZFN pair.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

V. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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1. A zinc three-finger binding domain that targets a nucleotide sequenceselected from the group consisting of GTGGAATTA (SEQ ID NO:1) andGAGTGGTTA (SEQ ID NO:2).
 2. The zinc three-finger binding domain ofclaim 1, where said zinc three-finger binding domain comprises asequence selected from SEQ ID NOS: 3-38 for GTGGAATTA (SEQ ID NO:1) andselected from the group consisting of SEQ ID NOS: 39-110 for GAGTGGTTA(SEQ ID NO:2).
 3. The zinc three-finger binding domain of claim 1,wherein said zinc three-finger binding domain comprises the nucleotidesequence of SEQ ID NO:116 for GTGGAATTA (SEQ ID NO:1) and the nucleotidesequence of SEQ ID NO:117 for GAGTGGTTA (SEQ ID NO:2).
 4. The zincthree-finger binding domain of claim 1, wherein said zinc three-fingerbinding domain is linked to a non-specific nuclease.
 5. The zincthree-finger binding domain of claim 4, wherein said non-specificnuclease is FokI.
 6. A zinc three-finger binding domain dimer thattargets a double-stranded nucleic acid comprising a first monomer thattargets nucleotide sequence GTGGAATTA (SEQ ID NO:1) in one strand and asecond monomer that targets nucleotide sequence GAGTGGTTA (SEQ ID NO:2)in the other strand.
 7. The zinc three-finger binding domain dimer ofclaim 6, wherein one monomer of said dimer comprises a sequence selectedfrom SEQ ID NOS: 3-5, 6-8, 9-11, 12-14, 15-17, 18-20, 21-23, 24-26,27-29, 30-32, 33-35 and 36-38 for GTGGAATTA (SEQ ID NO:1) and the othermonomer comprises a sequence selected from the group consisting of SEQID NOS: 39-41, 42-44, 45-47, 48-50, 51-53, 54-56, 57-59, 60-62, 63-65,66-68, 69-71, 72-74, 75-77, 78-80, 81-83, 84-86, 87-89, 90-92, 91-93,94-96, 97-99, 100-102, 103-105, 106-108, and 109-111 for GAGTGGTTA (SEQID NO:2).
 8. The zinc three-finger binding domain dimer of claim 6,wherein one monomer comprises the nucleotide sequence of SEQ ID NO:116for GTGGAATTA (SEQ ID NO:1) and the other monomer comprises thenucleotide sequence of SEQ ID NO:117 for GAGTGGTTA (SEQ ID NO:2).
 9. Thezinc three-finger binding domain dimer of claim 6, wherein each monomerof said zinc three-finger binding domain dimer is linked to anon-specific nuclease monomer.
 10. The zinc three-finger binding domaindimer of claim 9, wherein said non-specific nuclease is FokI.
 11. Thezinc three-finger binding domain dimer of claim 10, wherein said eachFokI nuclease monomer is different.
 12. A vector comprising a nucleicacid segment encoding a zinc three-finger binding domain that targets anucleotide sequence selected from the group consisting of GTGGAATTA (SEQID NO:1) and GAGTGGTTA (SEQ ID NO:2), said nucleic acid under thecontrol of a promoter operable in a eukaryotic cell.
 13. The vector ofclaim 12, further comprising a selectable or screenable marker.
 14. Thevector of claim 12, further comprising an origin of replication.
 15. Thevector of claim 12, wherein said vector is a viral vector.
 16. Thevector of claim 15, wherein said viral vector is an adenoviral vector,an adeno-associated viral vector, a pox viral vector, a herpes viralvector, a retroviral vector, a lentiviral vector.
 17. The vector ofclaim 16, wherein the lentiviral vector is an integrase-deficientvector.
 18. The vector of claim 12, wherein said vector comprises twonucleic acid segements, each encoding a zinc three-finger bindingdomain, one that targets GTGGAATTA (SEQ ID NO:1) and one that targetsGAGTGGTTA (SEQ ID NO:2).
 19. The vector of claim 18, wherein each ofsaid nucleic acid segments is under the control of a separate promoteractive in said eukaryotic cell.
 20. The vector of claim 18, wherein bothof said nucleic acid segments are under the control of a the samepromoter.
 21. The vector of claim 19, wherein said nucleic acid segmentsare separated by a transcription termination signal and/or apicornavirus T2A sequence.
 22. The vector of claim 20, wherein saidnucleic acid segments are separated by an internal ribosome entry site.23. A method of promoting recombination within a CTFR gene in a humancell comprising contacting said cell with a first zinc three-fingerbinding domain that targets a nucleotide sequence GTGGAATTA (SEQ IDNO:1) and a second zinc three-finger binding domain that targets anucleotide sequence GAGTGGTTA (SEQ ID NO:2), wherein each of said firstand second zinc three-finger binding domains are linked to anon-specific nuclease.
 24. The method of claim 23, wherein said firstzinc three-finger binding domain comprises a sequence selected from SEQID NOS: 3-5, 6-8, 9-11, 12-14, 15-17, 18-20, 21-23, 24-26, 27-29, 30-32,33-35 and 36-38 for GTGGAATTA (SEQ ID NO:1) and said second zincthree-finger binding domain comprises a sequence selected from the groupconsisting of SEQ ID NOS: 39-41, 42-44, 45-47, 48-50, 51-53, 54-56,57-59, 60-62, 63-65, 66-68, 69-71, 72-74, 75-77, 78-80, 81-83, 84-86,87-89, 90-92, 91-93, 94-96, 97-99, 100-102, 103-105, 106-108, and109-111 for GAGTGGTTA (SEQ ID NO:2)
 25. The method of claim 23, whereinsaid first zinc three-finger binding domain comprises the nucleotidesequence of SEQ ID NO:116 for GTGGAATTA (SEQ ID NO:1) and said secondzinc three-finger binding domain comprises the nucleotide sequence ofSEQ ID NO:117 for GAGTGGTTA (SEQ ID NO:2).
 26. The method of claim 23,wherein said human cell is a lung epithelial cell, and intestinalepithelial cell, a biliary duct epithelial cell, a gall bladderepithelial cell or pancreatic epithelial cell.
 27. The method of claim26, wherein said epithelial lung cell or pancreatic cell comprises aCFTR gene with a ΔF508 mutation.
 28. The method of claim 27, whereinsaid epithelial lung cell or pancreatic epithelial cell is located in aliving human subject.
 29. The method of claim 28, wherein contactingcomprises administering said first and second zinc three-finger bindingdomains to lung or pancreatic tissue of said subject.
 30. The method ofclaim 29, wherein administration to lung tissue comprises inhalation ortopical instillation.
 31. The method of claim 29, wherein administrationto pancreatic tissue comprises injection.
 32. The method of claim 23,wherein contacting comprises administering to said subject an expressionvector comprising a first nucleic acid segment encoding a first zincthree-finger binding domain that targets GTGGAATTA (SEQ ID NO:1) and asecond nucleic acid segment encoding a second zinc three-finger bindingdomain that targets GAGTGGTTA (SEQ ID NO:2), said nucleic acids underthe control of one or more promoters operable in a eukaryotic cell. 33.The method of claim 32, wherein said vector is a viral vector.
 34. Themethod of claim 33, wherein said viral vector is an adenoviral vector,an adeno-associated viral vector, a pox viral vector, a herpes viralvector, a retroviral vector, a lentiviral vector.
 35. The method ofclaim 34, wherein the lentiviral vector is an integrase-deficientvector.
 36. The method of claim of claim 32, wherein each of saidnucleic acid segments is under the control of a separate promoter activein said eukaryotic cell.
 37. The method of claim 32, wherein both ofsaid nucleic acid segments are under the control of a the same promoter.38. The method of claim 36, wherein said nucleic acid segments areseparated by a transcription termination signal.
 39. The method of claim37, wherein said nucleic acid segments are separated by an internalribosome entry site and/or a picornavirus T2A sequence.